Amplification of biotin-mediated targeting

ABSTRACT

The present invention relates to the delivery of drug, peptide and protein pharmaceuticals using a biotin-mediated uptake system. More particularly the invention relates to the amplification of active substance delivery with the biotin uptake system using a biotin-active substance-polymer conjugate or a biotin-nanoparticle conjugate. The invention also relates to processes for preparing the conjugates, pharmaceutical and diagnostic compositions containing same and methods of diagnosis and treatment involving the conjugates.

FIELD OF THE INVENTION

The present invention relates to the delivery of drug, peptide andprotein pharmaceuticals using a biotin-mediated uptake system. Moreparticularly the invention relates to the amplification of activesubstance delivery with the biotin uptake system using a biotin-activesubstance-polymer conjugate or a biotin-nanoparticle conjugate. Theinvention also relates to processes for preparing the conjugates,pharmaceutical and diagnostic compositions containing same and methodsof diagnosis and treatment involving the conjugates.

BACKGROUND OF THE INVENTION

In conventional cancer chemotherapy, to obtain a linear increase incancer-cells kill rates it is often necessary to exponentially increasethe dosage of cytotoxic drugs. This in turn leads to an undesirableincrease in non-specific cytotoxicity of bystander, healthy cells. Inorder to reduce the effect of the high dose of toxin on normal, healthytissues, it is often necessary to repeatedly deliver a smaller dose ofcytotoxin, which often leads to the survival of a small fraction ofdrug-resistant cells.

Attempts have been made to increase the dose of cytotoxic agentdelivered to the tumor cell, through the use of specific targetingagents such as monoclonal antibodies specific for “tumor-antigens”. Inmany cases it has been found that the resultant antibody-drug conjugateis highly immunogenic, and thus may lead to an antibody response againstthe conjugate, which means that treatment must be halted. For thisreason small, poorly immunogenic molecules, which have a highspecificity for tumour cells, have been sought as alternatives toantibody-mediated targeting agents.

Several candidate targeting agents have recently been identified, andthese agents include vitamins, which are essential for the growth ofrapidly dividing cells such as tumours. Two such vitamins, vitamin B12and folic acid, have been shown to target a small subset of aggressivetumour cell lines. Russell-Jones and co-workers have previouslydescribed the use of vitamin B12 and folic acid as targeting agents forthe delivery of polymers and nanoparticles containing, or linked to,pharmaceuticals both for oral delivery and also for cancer therapy (seefor example WO00/66090, WO00/66091 and WO94/27641).

However, the receptivity of cancer cells to these vitamins is variableas a consequence of differential upregulation of the cell-surfacereceptors for these vitamins. In particular, the aforementionedinventors have found, using in vitro studies involving numerous tumorcell-lines, that many tumor cells upregulate either vitamin B 12 orfolate receptors, but there are relatively few examples where receptorsfor both of these vitamins are simultaneously upregulated. The efficacyof vitamin B12 and folate-targeted chemotherapy is thus not optimal formany types of cancers. It would be desirable, therefore, to be able toutilize a targeting agent which could target a greater proportion oftumor cell-lines, to generate a targeted chemotherapy regime withbroader utility in the treatment of cancer.

Biotin is one of the water-soluble, B-group vitamins and is used forfat, protein and carbohydrate metabolism, cell growth and fatty acidproduction. Biotin has been employed in the laboratory as a trace and inimaging studies with IGG monoclonal antibodies. Biotin conjugates arereported in the literature, and many of the biotin conjugates of theprior art rely on biotin's very high affinity for avidin andstreptavidin. Biotin/avidin and biotin/streptavitin systems have beendeveloped for in vitro assay systems as well as in vivo targeting. Inthe latter case, biotin is bound to a targeting agent, such as anantibody or antibody fragment, which targets a specific area of thebody. Circulating biotin-conjugate is allowed to clear, or clearance isaccelerated through the use of another material. Finally, the materialto be targeted, be it a therapeutic or diagnostic agent, and which iscovalently linked to avidin or strepavidin, is administered, and thepowerful affinity of avidin or streptavidin for biotin ensures that ahigh proportion of the injected dose of the avidin/streptavidinconjugate is targeted to, and remains in, the region(s) of the bodycontaining the targeted biotin-conjugate.

Limitations exist in the use of biotin to target to tumor cells whenusing small-molecule, biotin-conjugate constructs (for example, onemolecule of drug for each molecule of biotin). The dose deliverable issmall because of the low receptor density, and, because of the smallsize of the biotin-drug conjugate, they are readily excreted in thekidneys and re-absorbed in the proximal tubules, where there is highdensity of biotin receptors. This leads to rapid removal of theconjugates from the circulation as well as undesirable accumulation ofbiotin-drug conjugates in the kidney. These limitations are demonstratedby prior art technology in U.S. Patent Appl No. 20020049154, whichteaches the use of biotin and other targeting agents to delivertherapeutic molecules within the body. The pharmaceutical constructsdisclosed are limited by size, and furthermore, only one molecule of theactive agent is covalently bonded to the biotin.

Cancer and related diseases remain a leading cause of death in today'ssociety. Accordingly there is a strong need to identify new, improved,better and/or alternative pharmaceutical compositions and agents for itstreatment, amelioration and prevention. There is a further need forchemotherapeutic agents which address some of the undesirable sideeffects of known agents. There is also a need for different therapies tobe available to physicians to combat the numerous and various types ofcancers and to provide new options for treatment to address issues oftolerance of proliferating cells to the existing chemotherapeutic agentsand treatment regimes. In addition there is a need for broad-spectrumchemotherapeutics in the field of cancer therapy.

Accordingly it is a preferred object of the present invention to providepharmaceutical and diagnostic compositions and methods for thediagnosis, treatment, amelioration or prophylaxis of disease by theamplification of active substance delivery to biological targets. Thepresent invention also seeks to provide diagnostic and pharmaceuticalcompositions and methods for targeting neoplastic cells for treatment,which compositions and methods provide improved cell activity in termsof targeting function and/or improved delivery of toxic and/ordiganostic agents.

SUMMARY OF THE INVENTION

Surprisingly, it has been found by the present inventors that biotinconjugates are able to act as targeting agents for the delivery ofmacromolecules to many biological targets associated with disease,including cancerous cells and tumours, sites of inflammation, andmacrophages and dendritic cells. The biotin conjugates of the inventionare large molecular weight complexes incorporating biotin or analoguesthereof and an active agent to be delivered. The biotin conjugates ofthe invention most preferably involve polymer or nanoparticle technologysuitable for the amplified delivery of the active agent

The invention further relates to the surprising observation that thevitamin, biotin, is able to target a much wider range of tumours thaneither vitamin B12 or folate. It is unexpected that biotin-drugconjugates would have such marked activity and wide application tobiological targets including cancerous cells and tumours, sites ofinflammation, and macrophages and dendritic-cells. This is becauseuptake of biotin is thought to occur through the sodium dependentmulti-vitamin transporter (SMVT), and consequently, while smallmolecules may be co-transported, large structures such as polymer-drugconjugates cannot be transported.

Simple conjugates of one targeting molecule with one molecule of anactive agent have significant drawbacks, for reasons discussed above.The above-mentioned limitations are addressed by incorporating manymolecules of the active agent (eg drug) within the conjugates of theinvention, such that the biotin targeting effect is amplified by theprovision of many more molecules of the active agent per biotin-receptorinteraction.

Conjugate-mediated amplification of the targeted drug delivery can beachieved either by attaching both the active agent and biotin (or biotinanalog) to a high molecular weight polymer, or incorporation of theactive agent within or on the surface of a nanoparticle, thenanoparticle being coated with biotin or an analogue thereof. Thus,amplification of active agent delivery can occur by a macromolecularconjugates such as a polymer or nanoparticle to which biotin (or ananalog) is attached in such a way that it is able to bind to biotinreceptors expressed on cell surfaces. Accumulation of the macromolecularbiotin-active agent conjugate in the kidneys is also minimised due tothe large size.

The biotin conjugates of the invention are particularly suitable forparenteral delivery to tumors as they can utilize the biotin receptorsystem for binding and uptake, and have the aforementioned advantage ofamplifying the amount of active agent which can be delivered via thebiotin uptake mechanism, as well as minimising or avoiding targeting tothe kidneys by virtue of their size. According to one aspect of theinvention there is provided a conjugate comprising at least one biotintargeting molecule or an analog thereof, in association with an activesubstance and a support for the amplified delivery of the activesubstance.

The conjugates of the invention preferably involve the use of polymersor nanoparticles as the support for the active substance andbiotin-targeting agent. Preferably, the nanoparticle is a nanosphere ora nanocapsule.

The conjugates of the invention comprise at least one targeting molecule(TM) which is a biotin molecule, or analogue thereof, wherein theability of the targeting molecule to undergo the binding reactionsnecessary for uptake and transport of biotin in a vertebrate host andthe activity of the active substance are substantially maintained,following incorporation and/or following biological release of theactive substance from the polymer, nanoparticle, or nanosphere.

Preferably, the biotin or biotin analogue is electrostatically orcovalently-linked to the polymer, or coats the surface of thenanoparticle. The active agents of the nanoparticle may be enclosed bythe nanoparticle or may coat the surface of the nanoparticle.

In a preferred embodiment of the invention the biotin-targeting moietyis in itself pharmaceutically active, such as by being cytotoxic orhaving anti-inflammatory activity.

The polymeric conjugates of the invention have the general formula:(B−Q)n−P−(Q′−A)m

wherein B is biotin, or an analogue thereof, which is a targetingmolecule which will bind to a surface biotin receptors on a cell, andwhere

-   -   n, the molar substitution ratio of B in the conjugate, is in the        range from 1.0 to 50.0;    -   P is a pharmaceutically acceptable linear, branched, or        dendritic polymer;    -   A is a diagnostic or pharmaceutically active substance;    -   m, the molar substitution ratio of A in the conjugate, is in the        range from 1.0 to 1000; and    -   Q and Q′ are independently a covalent bond, or a spacer compound        linking biotin, P and A by covalent bonds.

In a further aspect, there is provided a process for synthesising thepolymeric conjugates of the invention, comprising one or more of thefollowing steps:

-   -   a) reacting the active substance with the polymer to form said        conjugate:    -   b) chemically modifying the active substance to provide at least        one functional group capable of forming a chemical linkage, and        reacting the active substance and polymer to form said        conjugate:    -   c) chemically modifying the TM to provide at least one        functional group capable of forming a chemical linkage and        reacting the carrier and polymer to form said conjugate:    -   d) chemically modifying the active substance and the polymer to        provide functional groups capable of forming a chemical linkage,        and reacting the active substance and polymer to form said        conjugate:    -   e) reacting the active substance with at least one cross-linking        agent and reacting the active substance of polymer to form said        conjugate:    -   f) reacting the TM with at least one cross-linking agent and        reacting the polymer and TM to form said conjugate:    -   g) reacting the active substance and polymer with at least one        cross-linking agent and reacting the active substance and        polymer to form said conjugate:    -   h) reacting the active substance directly with a polymeric        support to form an intermediate containing one or more molecules        of the active substance linked to the polymer, and subsequently        coupling the polymer-active substance intermediate to one or        more targeting molecules:    -   i) coupling one or more TM molecules to a polymeric support and        subsequently reacting the carrier-polymer intermediate with one        or more molecules of the active substance to give a final        conjugate containing one or more molecules of the active        substance.

In another aspect of the invention there is provided a process for theproduction of a polymeric conjugate having the general formula:(B−Q)n−P−(Q′−A)m

wherein B, Q, P, Q′, A, n and m are as defined above, said processselected from:

-   -   a) reacting A with P to form an intermediate complex, and        thereafter reacting the intermediate conjugate with biotin;    -   b) reacting B with P to form an intermediate complex and        thereafter reacting the intermediate conjugate with A;    -   c) the process of step a) or step b) wherein one or more of B, P        or A are modified to provide at least one functional group        capable of forming a chemical linkage prior to coupling with the        other reactants; or    -   d) reacting one or two of B, P or A with Q and/or Q′ prior to        coupling with the other reactants.

In a further aspect of the invention there is provided a method for themodification of a polymeric support to introduce functional groupscapable of reacting either directly with the active substance or with achemically-modified form of the active substance. The resultingpolymer-active substance intermediate contains one or more molecules ofthe active substance, said intermediate being suitable for coupling tothe TM to give a conjugate capable of amplified delivery of the activesubstance.

The invention also provides a process for the production of thenanoparticle conjugates of the invention, comprising one or more of thefollowing steps:

-   -   (a) reacting nanospheres with biotin or a biotin analogue to        form the conjugate;    -   (b) chemically modifying the biotin molecule or analogue thereof        to provide at least one functional group capable of forming a        chemical linkage, and reacting nanospheres and the modified TM        to form the conjugate;    -   (c) reacting nanospheres with at least one cross-linking agent        to prepare “activated” nanoparticles which are reacted with a TM        to form the conjugate;    -   (d) reacting a TM with at least one cross-linking agent and        reacting the nanospheres with the reacted TM to form the        conjugate;    -   (e) reacting nanospheres and a TM with at least one        cross-linking agent to form the conjugate;    -   (f) reacting nanospheres with at least one cross-linking agent,        reacting a TM with at least one cross-linking agent and reacting        the reacted nanospheres and the reacted TM to form the        conjugate; or    -   (g) reacting a TM with at least one cross-linking agent to        prepare an analogue which is reacted with a hydrophobic moiety        to form a hydrophobic derivative of the TM; and then incubating        the hydrophobic derivative of the TM with the nanosphere in such        a manner that the nanosphere is coated hydrophobically with the        TM.

According to another aspect of the invention there is provided adiagnostic or pharmaceutical composition which comprises a conjugate ofthe invention in association with a pharmaceutically acceptable carrieror diluent.

According to another aspect of the invention there is provided a methodfor the treatment, prophylaxis or amelioration of disease, whichcomprises the step of administering to a subject a therapeuticallyeffective amount of a conjugate or composition of the invention.

In a preferred embodiment the disease is a form of cancer.

In a further preferred form, the disease is an inflammatory disease.

The conjugates of the invention can be used to stimulate macrophages anddendritic cells with antigens as the active agent through targeting ofthese complexes of biotin and antigen to biotin receptor positive cells.Moreover, the conjugates of the invention can be used to targetmacrophages with cytotoxic agents to reduce the severity ofmacrophage-mediated events in diseases such as psoriasis, colitis,Crohn's disease, multiple sclerosis, graft-versus-host reaction andrheumatoid arthritis.

Thus, according to another aspect of the invention there is provided amethod for stimulating macrophages or dendritic cells with an antigen bycontacting the macrophage or dendritic cell with a conjugate of theinvention, wherein the active agent is an antigen and the macrophage orcells to be contacted are biotin receptor positive.

In a further embodiment of the invention the conjugates can be used todeliver anti-parasitic drugs to macrophages. Such processes can be usedin the treatment of intracellular parasites such as malaria, salmonella,and leishmania.

In another embodiment of the invention, the conjugates can be used toenhance the transfer of the drug from the intestinal lumen to thebloodstream.

In a further embodiment, the invention provides a conjugate suitable forimaging of tumours or inflammatory conditions, the conjugate comprisingmore than one imaging agent linked to a polymer, or more than oneimaging agent which is incorporated within and/or coated on the surfaceof a nanosphere or nanoparticle, wherein the polymer, nanosphere ornanoparticle is linked to at least one targeting molecule which is abiotin molecule, or analogue thereof, wherein the ability of thetargeting molecule to undergo the binding reactions necessary for uptakeand transport of biotin in a vertebrate host and the activity of theimaging are substantially maintained, following incorporation and/orfollowing biological release of the active substance from the polymer,nanoparticle, or nanosphere.

Furthermore, the invention also provides a conjugate having a biotinmolecule, or analogue thereof, as a first targeting molecule, and one ormore second targeting molecules, wherein the ability of the first andsecond targeting molecules, individually or combined, provide thebinding reactions necessary for uptake and/or transport of biotin in acell and/or provide for release and/or promote a biological activity ofthe active substance in a cell.

According to another aspect of the invention there is provided the useof a conjugate of the invention in the manufacture of a medicament forthe diagnosis and/or treatment of disease.

According to another aspect of the invention there is provided the useof biotin or an analogue thereof in the manufacture of a conjugate ofthe invention.

According to another aspect of the invention there is provided an agentfor the diagnosis, treatment, prophylaxis or amelioration of a diseasewhich agent comprises a conjugate of the invention.

These and other aspects of the invention will become evident from thedescription and claims which follow, together with the accompanyingdrawings.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cryostat sections of P815 tumor cells taken from mice, 6hours post Rhodamine-HPMA injection, showing accumulation of thispolymer using fluorescent microscopy.

FIG. 2 shows an increased uptake of FITC fluorescent labelled polymersin ascites cells from L1210FR tumors using biotin as a target molecule.

FIG. 3 shows an increased uptake of FITC and TRITC fluorescent labelledpolymers in ascites cells from L1210FR tumors using biotin as a targetmolecule.

FIG. 4 shows an increased uptake of Rhodamine-HPMA polymer using biotinas target molecule in Ov2008 tumor cells.

FIG. 5 shows an increased uptake of Rhodamine-HPMA polymer using biotinas target molecule in RENCA tumor cells.

FIG. 6 shows an increased uptake of Rhodamine-HPMA polymer using biotinas target molecule in 4T1 tumor cells.

FIG. 7 shows an increased uptake of Rhodamine-HPMA polymer using biotinas target molecule in JC tumor cells.

FIG. 8 shows an increased uptake of Rhodamine-HPMA polymer using biotinas target molecule in MMTO60562 tumor cells.

FIG. 9 shows a growth of Colo-26 tumours following treatment withpolymer-linked doxorubicin (Dox).

FIGS. 10 a and 10 b show a plot of tumour growth following treatmentwith Dox-TP-HPMA-Colo-26. The data depicts mean.

DETAILED DESCRIPTION OF THE INVENTION

The conjugates of the present invention relate to a support to which anactive agent and a biotin molecule, or analogue thereof, are associatedor conjugated. These biotin complexes are directed to biological targetshaving an affinity for biotin, and are particularly suitable forparenteral delivery to tumours, cancerous cells, sites of inflammation,and to macrophages and dendritic cells. The conjugates of the inventionhave the advantage of increasing the amount of active agent which can bedelivered via a biotin uptake mechanism, as well as minimising oravoiding targeting to the kidneys by virtue of their size. The supportis preferably a polymer, nanoparticle, or nanosphere. Below are separatedescriptions for polymers and for nanoparticles/nanospheres:

Polymers

The polymer conjugates of the present invention are targeted to cancercells using biotin or analogues thereof as the targeting molecule. Oncethe drug-polymer conjugate has reached its target tissue, the conjugatebinds to a cell-surface receptor and initiates receptor-mediatedendocytosis, which transports the conjugate to the cell interior. Thependant drug may be released by the action of lysosomal enzymes, bycleavage of a disulfide linked drug by intracellular glutathione orotherwise. These polymeric conjugates may be used for oral delivery ofthe drug to the circulatory or lymphatic drainage system. Preferably,the polymeric conjugates and compositions of the invention relate totargeting the drugs/pharmaceuticals or imaging agents to sites ofdisease, especially tumor/cancer cells.

In a further embodiment the polymer conjugates of the present inventionhave been targeted to macrophages using biotin or analogues thereof asthe targeting molecule. Once the drug has reached an inflammatory site,the conjugate is endocytosed by the target macrophage and the pendantdrug may be released by the action of lysosomal enzymes, by cleavage ofa disulfide linked drug by intracellular glutathione, or by the acidenvironment within intracellular compartments such as endosomes andlysosomes, or other means.

While it is the belief of the inventors that the therapeutic benefitprovided by the polymer conjugates of this invention is provided by theabove stated mechanisms, it is possible that other mechanisms of actionmay provide benefit, and this invention is not restricted to any onemechanism of action.

The polymer, P (as defined above), of the present invention can be anypharmaceutically acceptable polymer. The polymer is able to attach to atleast one TM and to at least one, but preferably a multiplicity, ofactive substance molecules. The polymer may be naturally occurring orsynthetic or a mixture thereof, and can be linear, branched, ordendritic.

Suitable polymers for substitution with biotin and modificationaccording to the invention, include, but are not limited to,poly[N-(2-hydroxypropyl)-methacrylamide], dextran, chondroitan sulfate,water soluble polyurethanes formed by covalent linkage of PEG withlysine, poly(glutamic acid), poly(hydroxypropyl glutamine) and branchedchain polypeptides formed by the dual modification of the γ- and α-aminogroups of lysine during the peptide synthesis, as well as dendrimers andPEG-dendrimers, dextran, dextrin, glycosaminoglycans,carboxymethylcellulose, polylactic acid, polyglutamic acid,poly[lactide-co-glycolide], polyhydroxyethymethacrylate (poly-HEMA),human serum albumen (HSA), and other such biodegradable, ornon-biodegradable polymers. Such polymers may have multipleamino-termini, to which can be conjugated a plurality of thepharmaceutical or drug to be delivered. The polymers can also be formedwith multiple cystines, to provide free thiols, or multiple glutamatesor aspartates, to provide free carboxyls for conjugation using suitablecarbodiimides. Similarly the polymer can contain multiple histidines ortyrosines for conjugation. The polymer may have multiple hydroxyl groupssuitable for modification, or alternatively may contain vicinal hydroxylgroups suitable for oxidation with reagents such as periodic acid, suchthat chemistry well known in the art can be used to conjugate the TM anddrug. The polymer may also have multiple carboxy groups for conjugationusing suitable carbodiimides.

Preferably the linkage to the polymer, or the polymer to which thepharmaceutical is linked, should be degradable or biodegradable.Potentially biodegradable polymers include dextran and its derivatives,as well as dextrin, amino acid polymers such as polylysine,poly-glutamic acid, alginate, heparin sulphate, and other sulphatedpolysaccharides, gelatin, glycosaminoglycans,poly[lactide-co-glycolide], polyhydroxyethymethacrylate (poly-HEMA), HSAor other similar proteins.

Non-biodegradable polymers may also be employed in the present inventionand include poly[N-(2-hydroxypropyl)methacrylamide], to which isattached biodegradable side chains such as those containing esterlinkages, or amino acid sequences cleavable within lysosomal vacuolesie. Gly-Phe-Leu-Gly (Rihova, B. and J. Kopecek 1985 Biologicalproperties of targetablepoly[N-(2-hydroxypropyl)-methacrylamide]-antibody conjugates. J. ControlRel., 2:289-310]. Other amino acid spacers cleavable by intracellularproteases include Gly-Phe-Ala; Gly-Phe-Ala-Gly; Gly-Phe-Tyr-Ala; andGly-Phe-Tyr-Ala-Ala, Ala-Leu-Ala-Leu [Rejmanova, P., Obereigner, B., andKopecek, J. 1981 Makromol. Chem. 182:1899-1915].

The preferred TM is biotin, or an analogue of biotin, either of whichmay be adapted provided that binding to cell surface biotin receptors atdisease sites is still possible. Biotin is most easily covalentlyattached to a ligand, or the polymer, via its carboxylic acid moiety.Alternatively, the TM can be modified to have charged groups of oppositecharge to functional groups on the polymer such that the TM is bound bynon-covalent (electrostatic, H-bonded, and hydrophobic bonding) forces.

Suitable analogues of biotin, according to the invention include, butare not limited to biotin, iminobiotin, Biocytin hydrazide, Biotinhydrazide, biocytin, 5-(Biotinamido)pentylamine,Sulfo-NHS(n-Hydroxysuccinimidyl)-Biotin, Sulfo-HNS-hexanyl-biotin(Sulfo-NHS-LD-Biotin), NHS-Biotin, Pentafluorophenyl-biotin,Pentafluorophenyl-polyethylenoxide-biotin, NHS-biotinTrifluoroacetamide, NHS-Iminobiotin trifluoroacetamide,Maleimido-polyethylenoxide biotin, Maleimido-polyethylenoxideiminobiotin, desthiobiotin, chloracetyl-biotin.

Further biotin analogues include 3-(N-Maleimido-propionyl)biocytin:athiol-specific biotinylating reagent, alpha-dehydrobiotin, Z- andE-4,5-dehydrodethiobiotin, norbiotinamine, d1-4xi-(4-carboxybutyl)-5-carbethoxy-cis-hexahydropyrrolo(3,4-d)imidazol-2-one (N-carbethoxyazabiotin),d1-4xi-(2-carboxyethyl)-cis-hexahydropyrrolo-[3,4-d]imidazol-2-one(bisnorazabiotin), bis-allyloxycarbonyl biotin aldehyde, carboxybiotin,methyl biotin.

In one embodiment of the invention the linkage joining thepharmaceutical, or the biotin to the polymer is a disulfide bond. In afurther embodiment of the invention the linkage joining thepharmaceutical, or the biotin to the polymer is an ester linkage. In yetanother embodiment of the invention the linkage joining thepharmaceutical or the biotin to the polymer is a γ-glutamyl-ε-lysinebond. In yet another embodiment of the invention the linkage joining thepharmaceutical or the biotin to the polymer is a diazo-linkage. In yet afurther example the bond linking the drug to the polymer is an acidlabile linker, such as that formed with aconitic acid or via a hydrazonelinkage.

The spacer groups Q and Q′ are optional. When they are absent the biotinTM, and/or the active substance A are linked to polymer P by a directcovalent or electrostatic bond. Spacer groups are introduced either toimprove the biotin receptor affinity of the biotin conjugate or toovercome problems in the coupling of the carrier, biotin, and/or theactive substance A arising from unfavourable steric interactions betweenthe biotin and A with the polymer P, or to increase the bioactivity of Ain the conjugate. The spacer groups may also act as linking agents,being bi-functional compounds with selected functional groups on eachend to react with suitable functional groups located on the polymer, andalso on the biotin carrier molecule and/or on the pharmaceuticallyactive substances.

Suitable extended spacers for the conjugation of the pharmaceutical orbiotin to the polymer matrix include: disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BSS), ethyleneglycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (Sulfo-EGS), p-amino-phenylaceticacid, dithiobis(succinimidylpropionate) (DSP),3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidyltartarate (DST), disulfosuccinimidyl tartarate (Sulfo-DST),bis[2-(succinimidyloxycarbonyloxy)-ethylene]sulfone (BSOCOES),bis[2-(sulfosuccinimidooxycarbonyloxy)-ethylene]sulfone (Sulfo-BSOCOES),dimethyl adipimidate.2 HCl (DMA), dimethyl pimelimidate.2 HCl (DMP),dimethyl suberimidate.2 HCl (DMS),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl4-(p-maleimidophyl)butyrate (SMPB).

The active substance to be delivered is preferably a hormone, drug,prodrug, toxin, pharmaceutically active protein, immunogen, or DNA orRNA analogue.

Suitable toxins, according to the invention, include, but are notlimited to, ricin, abrin, diphtheria toxin, modecin, tetanus toxin,mycotoxins, mellitin, α-amanitin, pokeweed antiviral protein, ribosomeinhibiting proteins, especially those of wheat, barley, corn, rye,gelonin, maytansinoid.

Suitable cytotoxic agents, according to the invention, include, but arenot limited to alkylating agents such as chlorambucil, cyclophosphamide,melphalan, cyclopropane; anthracycline antitumor antibiotics such asdoxorubicin, daunomycin, adriamycin, mitomycin C,[2-(hydroxymethyl)anthraquinone]; antimetabolites such as methotrexate,dichloromethatrexate: cisplatin, carboplatin, and metallopeptidescontaining platinum, copper, vanadium, iron, cobalt, gold, cadmium,gallium, iron zinc and nickel. Other agents include DON, thymidine,pentamethylmelamin, dianhydrogalactitol, 5-Methyl-THF, anguidine,maytansine, neocarzinostatin, chlorozotocin, AZQ, 2′deoxycoformycin,PALA, AD-32, m-AMSA and misonidazole, deferoxamine, ferrioxamine,iron-basic porphine.

Additional cytotoxins which may be employed in the conjugates of theinvention include epirubicin, platinum derivatives, includingcis-Platin, CarboPlatin, oxaliplatin, multinuclear platinate speciesincluding BBR3464 and BBR3005, transdiamminedichloroplatinum (II)(Transplatin), chlorodiethylenetriammineplatinum (II), Platinum IVcompounds, spiroplatin, platin-phosphine derivatives, calicheamycin,dolastatin derivatives, including auristatin, monomethylauristatin.

Suitable imaging agents, according to the invention include, but are notlimited to those described by Molecular Probes (Handbook of fluorescentprobes and research products) included by way of reference), such asRhodamine, fluorescein, Texas red, Acridine Orange, Alexa Fluor(various), Allophycocyanin, 7-aminoactinomycin D, BOBO-1, BODIPY(various), Calcien, Calcium Crimson, Calcium green, Calcium Orange,6-carboxyrhodamine 6G, Cascade blue, Cascade yellow, DAPI, DiA, DiD,Dil, DiO, DiR, ELF 97, Eosin, ER Tracker Blue-White, EthD-1, Ethidiumbromide, Fluo-3, Fluo4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258,Hoechst 33342, 7-hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye,Lissamine rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167,LysoSensor Green, LysoSensor Yellow/Blu, Lysotracker Green FM, MagnesiumGreen, Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos,MitoTracker Red CMXRos, Monobromobimane, NBD amines, NeruoTrace 500/525green, Nile red, Oregon Green, Pacific Blue. POP-1, Propidium iodide,Rhodamine 110, Rhodamine Red, R-Phycoerythrin, Resorfin, RH414, Rhod-2,Rhodamine Green, Rhodamine 123, ROX dye, Sodium Green, SYTO blue(various), SYTO green (Various), SYTO orange (various), SYTOX blue,SYTOX green, SYTOX orange, Tetramethylrhodamine B, TOT-1, TOT-3,X-rhod-1, YOYO-1, YOYO-3.

Additionally radionuclides can be used according to the invention eitheras imaging agents or as pharmaceutically active substances. Theseradionuclides include, but are not limited to radioactive species ofFe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(I1) Indium, Gallium,Technetium, such as ^(99m)Technetium. ¹¹¹Indium, ¹⁸⁶Re, ¹⁸⁶Re^(66,67,68)Ga, ⁹⁰Y ¹⁴⁹Pm, ¹⁷⁷Lu, ²⁷Mg, ⁴⁷Ca, ⁶⁴Cu. Also are includedmetal ions generally used for chelation in paramagnetic T1-type MIRcontrast agents, and include di- and tri-valent cations selected fromthe group consisting of copper, chromium, iron, gadolinium, manganese,erbium, europium, dysprosium and holmium. Metal ions that can bechelated and used for radionuclide imaging according to the invention,include, but are not limited to metals selected from the groupconsisting of gallium, germanium, cobalt, calcium, indium, iridium,rubidium, yttrium, ruthenium, yttrium, technetium, rheniumn, platinum,thallium and samarium. Additionally metal ions known to be useful inneutron-capture radiation therapy include boron and other metals withlarge nuclear cross-sections. Also included are metal ions useful inultrasound contrast, and X-ray contrast compositions.

Suitable metal chelators according to the invention include, but are notlimited to HYNIC (2-hydrazinonicotinic acid), HYBIN, DTPA(N-diethylenetriaminopentaacetic acid), cyclams, DOTA and itsderivatives (1,4,7,10-tetraazacyclododecane- N,N′,N″, N″′-tetraaceticacid), TETA. TETA(1,4,8,11-tetraazacyclotetradecane-1,4,8,-11-tetraacetic acid), NOTA.NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid),

Suitable cross-linking agents for use in the preparation ofthiol-cleavable biodegradable linkers include N-succinimidyl3-(2-pyridyldithio)propionate (SPDP), iminothiolane, sulfosuccinimidyl6-[3-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP),succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (LC-SPDP),sulfosuccinimidyl 6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate(Sulfo-LC-SMPT), 1,4-di[3′-(2′-pyridyldithio)propionamido]butane(DPDPB), 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene(SMPT), dimethyl 3,3′dithiobispropionimidate.2 HCl (DTBP).

Additional linkers include those consisting of or containing5-benzoyl-valeric acid, valine-citrilline dipeptide,phenylalanine-lysine dipeptide, Gly-Phe-Leu-Gly.

It is within the scope of this invention to deliver other activesubstances or utilize other linkers known in the art.

Furthermore, it is within the scope of this invention to deliver two ormore different active substances by attaching said two or more activecompounds to the polymer by the methods described above.

Furthermore, it is within the scope of this invention to utilize one ormore TM in addition to biotin (or a biotin analog) by attaching to thepolymer the two (or more) different TMs. Additional TMs include, but arenot limited to, vitamin B12 and folic acid (and folic acid derivatives).

Nanaoparticles and Nanospheres

Two basic forms of nanoparticles have been developed, nanocapsules (ormicrocapsules) and nanospheres (or nanospheres), for enclosing, holdingor containing an active substance. The terms “nanoparticle”,“nanocapsule”, and “nanosphere” as used throughout the specificationrefer to a material or construct ranging in size from 1 nanometer to 100micrometers in size, which may be spherical or have some other shape.

The nanoparticle conjugates of the present invention have been targetedto cancer cells using biotin or analogues thereof as the targetingmoiety. The drug may be released from the nanoparticle to thecirculatory or lymphatic drainage system, and most preferably to thetarget tissue of the host. Whilst it is possible that these nanoparticleconjugates could be used for oral delivery of the drug to thecirculatory or lymphatic drainage system in general, the products ofthis invention preferably relate to targeting the drugs, pharmaceuticalsto the sites of disease, especially tumor/cancer cells.

The active substance to be delivered is preferably a hormone, drug,prodrug, toxin, pharmaceutically active protein, immunogen, or DNA orRNA analogue.

In essence the nanoparticles can be formed by any number of methods,several of which are outlined below:

(i) Solvent Evaporation

In which a compound which is soluble in one solvent is dispersed intoanother miscible solvent and the first solvent is evaporated off.Particles formed in this fashion have been used to administer(parenterally) a number of water insoluble compounds. An example of sucha system would be the formation of polyalkylcyanoacrylate nanocapsulesin which the anticancer agent, 5-fluorouracil is entrapped.

(ii) Desolvation

In this method a compound is contained in a liquid in which it issoluble (the solvent) and a second liquid (which is miscible with thefirst liquid, but in which the compound is not soluble) is added to thesolvent. As more of the second liquid is added the compound becomesdesolvated. During the process of desolvation the compound rich phase(the coacervate) contains an enriched amount of compound which isdispersed as microdroplets in the compound deficient phase. At thisstage the coalesced material can be chemically crosslinked by a suitablecrosslinking agent to form micro- or nano-particles. Nanoparticles ofgelatin or BSA can be prepared in this way. Solutions of these proteinsare dessolvated by the addition of sodium sulfate, or ammonium sulfatesolutions. At the point of desolvation there is an increase inturbidity, at which time the nanoparticles can be formed by the additionof a suitable cross-linker such as glutaraldehyde or butanedione.Alternatively a biodegradable cross-linker could be employed, such as alinker containing a disulfide bond, an azo-bond, or an esterasecleavable bond.

(iii) Complex Coacervation

In this procedure two polyelectrolytes having opposite charge are mixedin aqueous medium so that a spontaneous liquid/liquid phase separationoccurs. The phenomenon is limited to polymers having a suitable ioniccharge density and chain length. Typically these nanospheres are formedby the addition of a polyanion such as Gum Arabic, Alginate, orPolyphosphate, to a polycation such as Gelatin. Suitable particles arereadily formed by the complexation of gelatin and carboxymethylcellulose. The rate of release of pharmaceutical from such complexes canbe controlled by the addition of a suitable cross-linker such asglutaraldehyde or butanedione. Alternatively a biodegradablecross-linker could be employed, such as a linker containing a disulfidebond, an azo-bond, or an esterase cleavable bond.

(iv) Polymer/Polymer Incompatability

This procedure is based upon the observation that two chemicallydifferent polymers dissolved in a common solvent are usuallyincompatible. Thus the mixture will tend to form two phases. Theinsoluble phase can be used to coat core particles to formmicrocapsules. An example would be the precipitation of ethyl cellulosefrom cyclohexane by the addition of polyethylene.

(v) Interfacial Polymerization

In this technique, two reactants, each dissolved in a mutuallyimmiscible liquid, diffuse to the interface between the two liquidswhere they react to form a capsule wall. An example of such capsuleformation would occur if a mixture of Sebacoyl chloride dissolved in anoil phase and emulsified into an aqueous phase containingethylenediamine.

Other methods of formation of nanoparticles, nanocapsules, andnanospheres are known in the art, and can be applied for the purpose ofconstructing nanoparticles for the present invention

In one embodiment, the invention provides a conjugate between biotin anda biodegradable nanosphere in which is trapped a toxin or cytotoxicagent or active substance.

Suitable analogues of biotin, according to the invention include, butare not limited to biotin, iminobiotin, Biocytin hydrazide, Biotinhydrazide, biocytin, 5-(Biotinamido)pentylamine,Sulfo-NHS(n-Hydroxysuccinimidyl)-Biotin, Sulfo-HNS-hexanyl-biotin(Sulfo-NHS-LD-Biotin), NHS-Biotin, Pentafluorophenyl-biotin,Pentafluorophenyl-polyethylenoxide-biotin, NHS-biotinTrifluoroacetamide, NHS-Iminobiotin trifluoroacetamide,Maleimido-polyethylenoxide biotin, Maleimido-polyethylenoxideiminobiotin, Iodoacetyl-biotin, Chloroacetyl-biotin.

Suitable toxins, according to the invention, include, but are notlimited to, ricin, abrin, diphtheria toxin, modecin, tetanus toxin,mycotoxins, mellitin, alpha-amanitin, pokeweed antiviral protein,riosome inhibiting proteins, especially those of wheat, barley, corn,rye, gelonin, maytansinoid.

Suitable cytotoxic agents, according to the invention, include, but arenot limited to alkylating agents such as chlorambucil, cyclophosphamide,melphalan, cyclopropane; anthracycline antitumor antibiotics such asdoxorubicin, daunomycin, adriamycin, mitomycin C,[2-(hydroxymethyl)anthraquinone]; antimetabolites such as methotrexate,dichloromethatrexate: cisplatin, carboplatin, and metallopeptidescontaining platimun, copper, vanadium, iron, cobalt, gold, cadmium,iron, gallium, zinc and nickel. Other agents include DON, thymidine,pentamethylmelamin, dianhydrogalactitol, 5-Methyl-THF, anguidine,maytansine, neocarzinostatin, chlorozotocin, AZQ, 2′deoxycoformycin,PALA, AD-32, m-AMSA and misonidazole.

Polymers suitable for the formation of nanospheres by solventevaporation (in liquid drying) include, amongst others, Poly-lacticacid, Poly-(Lactide/co-glycolide), Poly-hydroxybutyrate,Poly-hydroxyvalerate, Poly-(hydroxybutyrate/valerate), Ethyl cellulose,Dextran, Dextrin, Polysaccharides, Polyalkylcyanoacrylate,Poly-methyl-methacrylate, poly(e-caprolactone) and various combinationsand co-polymers of the above.

Polymers suitable for the formation of nanospheres by interfacialprecipitation/polymerization include, amongst others, EUDRAGITTM;Poly(N′,N″L-lysinediylterephthaloyl); polymers formed by the reaction ofLysine hydrochloride and p-phthaloyl dichloride; by the reaction ofacryloylated maltodextrin or acryloylated hydroxyethyl starch withammonium peroxodisulfate and N,N,N′,N′-tetrarnethylethylenediamine.Nanospheres can also be formed by the polymerization of various diaminessuch as ethylene diamine, phenylenediamine, toluene diamine,hexamethylene diamine, or diols such as ethylene diol, bisphenol,resorcinol, catechol, pentanediol, hexanediol, dodecanediol, 1,4butanediol, with diacid chlorides such as sebacoylchloride and adipoylchloride, or diisocynates such as hexamethylene diisocyanate using themethods fully described in EPA 85870002.4.

Polymers suitable for the formation of nanospheres by polymer phaseseparation include co-poly(vinyl chloride:vinyl alcohol:vinyl acetate),cellulosic polymers, polyvinyl acetate, polyvinyl alcohol,polyvinylchloride, natural and synthetic rubbers, polyacrylates,polystyrene and the like. Methods to synthesize such nanospheres arefully described in U.S. Pat. No. 4,166,800.

Polymers suitable for the formation of nanospheres by complexcoacervation include, amongst others, mixtures of polyanions, such asgum arabic, alginate, carboxymethyl cellulose, carboxymethyl starch,polystyrene sulfonic acid, polyvinyl sulfonic acid, poly-D-glucuronicacid, Poly-pyruvic acid, carrageenan, heparin sulphate, polyphosphatewith polycations, such as polylysine, gelatin.

Polymers suitable for the formation of nanospheres by Polymer/Polymerincompatability include, amongst others, ethyl cellulose, Ethylene vinylacetate polymer, Poly(lactide), or Poly(vinylidene chloride) mixed withpolymers such as Polyethylene, Silicone, Polyisobutylene orPolybutadiene.

Other materials suitable for formation of nanospheres include, Starch,Cross-linked Albumen, Polyacrylamide, Cross-linked gelatin and othersobvious to those skilled in the art of nanosphere preparation.

The cross-linking agent may contain a disulfide bond or be cleavable byacid, base or periodate. Examples of suitable cross-linking agentsinclude: N-(4-azidophenylthio)phthalimide; 4,4′-dithiobisphenylazide;dithiobis(succinimidylpropionate);dimethyl-3,3′-dithiobispropionimidate.2HCl;3,3′-dithiobis-(sulfosuccinimidylpropionate);ethyl-4-azidophenyl)-1,3′dithiopropionate;sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiobutyrimuidate.HCl;N-succinimidyl-(4-azidophenyl)-1,3′dithiopropionate;sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate;sulfosuccinimidyl-2-(p-azidosalicylamido)-ethyl-1,3′-dithiopropionate;N-succinimidyl-3-(2-pyridylthio)propionate;sulfosuccinimidyl-(4-azidophenyldithio)-propionate; 2-iminothiolane;disuccinimidyl tartrate; andbis-[2-(succinimidyloxycarbonyloxy)-ethyl]-sulfone.

Suitable linking of the TM to the nanospheres may be achieved byreaction of the TM with a carbodiimide and N-hydroxysuccinimide (NHS),and then reacting the NHS derivative with a suitable functional group onthe nanosphere.

Examples of pharmaceutically acceptable carriers, diluents andexcipients for oral delivery include sodium bicarbonate solutions andsimilar diluents which neutralise stomach acid or have similar bufferingcapacity, glycols, oils or emulsions; and include medicaments in theform of gels, pastes and viscous colloidal dispersions. The medicamentmay be presented in capsule, tablet, slow release or elixir form or as agel or paste. Furthermore the medicament may be presented as a food.

Examples of pharmaceutically acceptable carriers, diluents andexcipients for parenteral delivery include saline, glycols, oils oremulsions; and include medicaments in the form of gels, pastes andviscous colloidal dispersions. It is within the scope of this inventionto incorporate the active substance within the nanoparticle and/or tocoat the active substance on the surface of the particle, provided thatthe TM bound to the surface of the nanoparticle is available forreceptor-binding to cell-surface biotin receptors at the sites ofdisease.

Furthermore, it is within the scope of this invention to attach the TMto the nanoparticle either by covalent bonding, or by physical coating,in which the TM is bound by a combination of electrostatic, H-bondingand/or hydrophobic bonding.

Furthermore, it is within the scope of this invention to deliver otheractive substances or utilize other material (from that described above)known in the art for the formation of nanoparticles.

Furthermore, it is within the scope of this invention to deliver two ormore different active substances by incorporating and/or coating saidtwo (or more) active compounds within and/or onto the nanoparticle bythe methods described above.

Furthermore, it is within the scope of this invention to utilize one ormore TM in addition to biotin (or a biotin analog) by attaching to thenanoparticle the two (or more) different TMs. Additional TMs include(but are not limited to) vitamin B12 and folic acid (and folic acidderivatives).

The compositions described herein, when used for the treatment ofdisease, may conceivably be used with or without the use of otherpharmaceutical agents.

Compositions have been described herein possessing a singlepharmaceutically-active ingredient, either attached or incorporated. Itis within the scope of this invention for compositions to possess aplurality of pharmaceutically-active compounds, their derivatives and/orprodrugs, either attached or incorporated, such combinations ofpharmaceutically-active compounds providing an additive or synergisticbenefit in the treatment of disease.

The terms “conjugate” and “macromolecular conjugate” are used herein intheir broadest sense to include all forms and synthetic stages (ieintermediate conjugates) of the biotin-mediated targeting compounds,compositions, complexes of the invention.

As used herein, the terms “treatment”, “prophylaxis” or “prevention”,“amelioration” and the like are to be considered in their broadestcontext. In particular, the term “treatment” does not necessarily implythat an animal is treated until total recovery. Accordingly, “treatment”includes amelioration of the symptoms or severity of a particularcondition or preventing or otherwise reducing the risk of developing aparticular condition.

The amount of the conjugate of the invention which is required in atherapeutic treatment according to the invention will depend upon anumber of factors, which include the specific application, the nature ofthe particular compound used, the condition being treated, the mode ofadministration and the condition of the patient. The conjugates may beadministered in a manner and amount as is conventionally practised. Thespecific dosage utilised will depend upon the condition being treated,the state of the subject, the route of administration and other wellknown factors as indicated above. The length of dosing may range from asingle dose given once every day or two, to twice or thrice daily dosesgiven over the course of from a week to many months to many years asrequired, depending on the severity of the condition to be treated oralleviated. It will be further understood that for any particularsubject, specific dosage regimens should be adjust over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions.

The production of pharmaceutical compositions for the treatment of thetherapeutic indications herein described are typically prepared byadmixture of the conjugates of the invention with one or morepharmaceutically or veterinary acceptable carriers and/or excipients asare well known in the art.

Examples of pharmaceutically acceptable carriers, diluents andexcipients for oral delivery include sodium bicarbonate solutions andsimilar diluents which neutralise stomach acid or have similar bufferingcapacity, glycols, oils or emulsions; and include medicaments in theform of gels, pastes and viscous colloidal dispersions. The medicamentmay be presented in capsule, tablet, slow release or elixir form or as agel or paste. Furthermore the medicament may be presented as a food.Examples of pharmaceutically acceptable carriers, diluents andexcipients for parenteral delivery include saline, glycols, oils oremulsions; and include medicaments in the form of gels, pastes andviscous colloidal dispersions.

In particular, the carrier must, of course, be acceptable in the senseof being compatible with any other ingredients in the formulation andmust not be deleterious to the subject. The carrier or excipient may bea solid or a liquid, or both, and is preferably formulated with thecompound as a unit-dose, for example, a tablet, which may contain up to100% by weight of the active compound, preferably from 0.5% to 59% byweight of the active compound. One or more active compounds may beincorporated in the formulations of the invention, which may be preparedby any of the well known techniques of pharmacy consisting essentiallyof admixing the components, optionally including one or more accessoryingredients. The preferred concentration of active compound in the drugcomposition will depend on absorption, distribution, inactivation, andexcretion rates of the drug as well as other factors known to those ofskill in the art.

The formulations of the invention include those suitable for oral,rectal, optical, buccal (for example, sublingual), parenteral (forexample, subcutaneous, intramuscular, intradermal, or intravenous) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulation suitable for oral administration may be presented indiscrete units, such as capsules, sachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture such as to form a unit dosage. Forexample, a tablet may be prepared by compressing or moulding a powder orgranules containing the active compound, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the compound of the free-flowing,such as a powder or granules optionally mixed with a binder, lubricant,inert diluent, and/or surface active/dispersing agent(s). Mouldedtablets may be made by moulding, in a suitable machine, the powderedcompound moistened with an inert liquid binder.

Formulations suitable for buccal (sublingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Compositions of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theconjugates of the invention, which preparations are preferably isotonicwith the blood of the intended recipient. These preparations arepreferably administered intravenously, although administration may alsobe effected by means of subcutaneous, intramuscular, or intradermalinjection. Such preparations may conveniently be prepared by admixingthe compound with water or a glycine buffer and rendering the resultingsolution sterile and isotonic with the blood. Injectable formulationsaccording to the invention generally contain from 0.1% to 60% w/v ofactive compound and are administered at a rate of 0.1 ml/minute/kg.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing theconjugates with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations or compositions suitable for topical administration to theskin preferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol, or oil. Carriers which may be used include Vaseline,lanoline, polyethylene glycols, alcohols, and combination of two or morethereof. The active compound is generally present at a concentration offrom 0.1% to 5% w/w, more particularly from 0.5% to 2% w/w. Examples ofsuch compositions include cosmetic skin creams.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 M to 0.2 M concentration with respect tothe said active compound. See for example Brown, L., et al. (1998).

Formulations suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Panchagnula R, et al.,2000) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2M active ingredient.

Formulations suitable for inhalation may be delivered as a spraycomposition in the form of a solution, suspension or emulsion. Theinhalation spray composition may further comprise a pharmaceuticallyacceptable propellant such as carbon dioxide or nitrous oxide.

The conjugates may be provided in the form of food stuffs, such as beingadded to, admixed into, coated, combined or otherwise added to a foodstuff. The term food stuff is used in its widest possible sense andincludes liquid formulations such as drinks including dairy products andother foods, such as health bars, desserts, etc. Food formulationscontaining compounds of the invention can be readily prepared accordingto standard practices.

Therapeutic methods, uses and compositions may be for administration tohumans or animals, including mammals such as companion and domesticanimals (such as dogs and cats) and livestock animals (such as cattle,sheep, pigs and goats), birds (such as chickens, turkeys, ducks), fishand other marine organisms, and the like.

The conjugates or pharmaceutically acceptable derivatives, for exampleprodrugs or salts thereof, can also be co-administered with other activematerials that do not impair the desired action, or with materials thatsupplement the desired action, such as antibiotics, antifungals,antiinflammatories, or antiviral compounds. The conjugates can comprisefurther drugs in combination or as a synergistic mixture.

The co-administration may be simultaneous or sequential. Simultaneousadministration may be effected by the compounds being in the same unitdose, or in individual and discrete unit doses administered at the sameor similar time. Sequential administration may be in any order asrequired and typically will require an ongoing physiological effect ofthe first or initial active agent to be current when the second or lateractive agent is administered, especially where a cumulative orsynergistic effect is desired.

Without being limited to any one mode or principle, it is postulatedthat upregulation of a biotin receptor other than the sodium dependentmulti-vitamin transporter (SMVT) might be responsible for the efficacyof the conjugates of the invention. It is generally accepted that uptakeof biotin occurs through the SMVT, which permits co-transport of onlysmall molecules, whose size is considerably less than that of theconjugates of the invention. This suggests that uptake ofconjugate-bound biotin may be due to another different biotin bindingsurface protein/receptor, working in collaboration or independentlyfrom, the SMVT. The inventors have also found that the intracellularfate of biotin, once internalized, is different from either vitamin B12or folate. As such, the intracellular processing of biotin-drugconjugates may be different from both Vitamin B12- or folate-targetedconjugates. This receptor profile and/or intracellular processing maythus contribute to one or more improved properties of the conjugates ofthe invention.

The present invention is further described with reference to thefollowing examples which are in no way limiting on the scope of theinvention.

EXAMPLES Example 1 Synthesis of Multi-Lysine Polymer 1 (MLP1)

A multi-lysine polymer (MLP1) of the formula[(NH₂-Gly)₄-Lys₂-Ser₂-Lys]₅-Ala-COOH, was synthesized on an AppliedBiosystems peptide synthesiser. More precisely this represents[(NH₂-Gly)₄-Lys₂-Ser₂-Lys]₄[Gly₄-Lys₂-Ser₂-Lys]-Ala-COOH

The formula [(NH₂-Gly)₄-Lys₂-Ser₂-Lys]₄[Gly₄-Lys₂-Ser₂-Lys]-Ala-COOH canbe represented as follows:

which show the structure more precisely.

Example 2 Synthesis of Multi-Lysine Polymer 2 (MLP2)

A multi-Lysine polymer (MLP2) of the general formula[(NH₂-Gly)₁₆-Lys₈-Lys₄-His₄-Glu₄-Lys₂-Lys]-Gly₅-Cys-COOH was synthesizedon an Applied Biosystems peptide synthesiser. More precisely thestructure can be represented as follows:

Example 3 Preparation of NHS-Biotin

Biotin (5 g) was dissolved in 100 ml dry dimethyl sulfoxide (DMSO), plus2.5 ml triethylamine.

N-hydroxysuccinimide (2.6 gm) was added as a powder to the biotin andreacted overnight with 4.7 gm dicyclohexylcarbodiimide at roomtemperature. The dicyclohexylurea was removed by filtration. The DMSOwas concentrated under reduced pressure and heating, and NHS-biotinprecipitated with diethylether.

The product was washed several times with anhydrous ether, dried undervacuum and stored as a white powder.

Example 4 Formation of MLP-toxin conjugates using biodegradablecross-linkers.

There are many toxins which could be used for formation ofbiotin-MLP-toxin conjugates, including momordin, Pseudomonas exotoxin A,ricin and abrin. A general method for the formation of biotin-MLP-toxinconjugates is described below:

Conjugates are prepared in which the covalent linker contains abiodegradable disulfide bond, which would be reduced in vivo, presumablyby intracellular glutathione in the tumor cell, thereby releasing theactive substance after transport from the serum into the tumor cell.Briefly, MLP1 or MLP2 was reacted with N-succinimidyl3-(2-pyridyldithio)propionate (SPDP). The dithiopyridyl-MLP (DTP-MLP)product was purified by RP-HPLC. A free thiol was introduced onto thetoxin by a two step procedure in which the toxin was firstly reactedwith SPDP, after which the thiopyridyl group was reduced withmercapto-ethanol. The product was purified by RP-HPLC. Alternativelyfree thiol was introduced into the toxin directly by reaction withiminothiolane. The thiolated product (SH-HN⁺toxin) was purified byRP-HPLC. Formation of the disulfide linked MLP-toxin conjugates wasachieved by reaction of the thiolated toxin derivative with DTP-MLP in2.5% acetic acid for 24 hours. The conjugated material was purified bySephadex G-25 chromatography, followed by RP-HPLC.

Example 5 Preparation of Poly-Drug-HPMA-Biotin Conjugate.

Two N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers were synthesizedas polymer backbones for the incorporation and derivatization withcytotoxic drugs and biotin. A non-biodegradable polymer backbone(HPMA-GG) was synthesized by the free radical copolymerization of HPMAwith N-methacryloylglycylglycine p-nitrophenyl ester. A biodegradablepolymer (HPMA-GFALG) was synthesized by the free radicalcopolymerization of HPMA withN-methacryloylglycylphenylalanylleucylglycine p-nitrophenol ester by themethod of Rejmanova and co-workers [Rejmanova, P., Obereigner, B., andKopecek, J. 1981 Makromol. Chem. 182:1899-1915]. In order to incorporatericin A chain and biotin onto the polymers, they were reacted with a tenmolar excess of a mixture of aminohexyl-biotin andDithiopyridyldodecylsuberyl-hexylamine (1:10 mole:mole) overnight.Unreacted nitrophenyl esters were subjected to aminolysis by theaddition of 1-amino-2-propanol. The modified polymers were purified bychromatography on Sepharose 6B. A solution of thedithiopyridyldodecylsuberylhexyl modified biotin-substituted polymerswas dissolved in 2.5% acetic acid and reacted with ricin A chain. Thereaction mixture was left for 144 hours at 4° C., afterwhich thericin-biotin-substituted polymers were purified by chromatography onSepharose 6B.

Example 6 Preparation of Poly-Daunomycin-HPMA-Biotin Conjugate

A N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer was synthesized asa polymer backbone for the incorporation and derivatization with boththe cytotoxic drug, daunomycin and biotin. A biodegradable polymer(HPMA-GFLG) was synthesized by the free radical copolymerization of HPMAwith N-methacryloylglycylphenylleucinylglycine p-nitrophenol ester bythe method of Rejmanova and co-workers [Rejmanova, P., Obereigner, B.,and Kopecek, J. 1981 Makromol. Chem. 182:1899-1915]. In order toincorporate daunomycin and biotin onto the polymers, they were reactedwith a ten molar excess of a mixture of aminohexyl-biotin and daunomycin(1:10 mole:mole) overnight. Unreacted nitrophenyl esters were subjectedto aminolysis by the addition of 1-amino-2-propanol. The modifiedpolymers were purified by chromatography on Sepharose 6B.

Example 7 Preparation of ¹²⁵I Labelled Polymers

Bolton-Hunter reagent was dissolved at 1 mg/ml in DMSO. Theamino-derivatized polymer was dissolved at 5 mg/ml in DMSO or DWcontaining 25 μl/ml DIEA. A 3 μl aliquot of Bolton-Hunter was added to20 μl of the polymer solution. The reaction was allowed to proceed for 3hours. Unreacted Bolton-Hunter was extracted with DCM (5×100 μl) afteraddition of 50 μl water. ¹²⁵I(1 μl) was added to the derivatizedpolymer, followed by the addition of 4 μl Chloramine-T dissolved at 20mg/ml in PBS. The reaction proceeded for 15 secs, at which time theradioactive polymer was purified on PD10 column which had beenequilibrated with 2.5% AcOH.

Example 8 Alternative Method of Preparation ofHydroxypropylmethacrylamide (HPMA)

1-Amino-2-propanol (58 g) was dissolved in acetonitrile (225 ml). Thesolution was cooled to −10° C. using an ethanol/dry ice bath.Methacryloyl chloride (40 g) in acetonitrile (170 ml) was added dropwisewith vigorous stirring from a pressure equalising dropping funnel. Themixture was then allowed to warm slowly to room temperature overnight.The hydrochloride salt of 1-amino-2-propanol was removed by filtrationthrough Celite filter aid. The solvent was removed at reduced pressurewith a bath temperature of 50° C. The product was isolated by dissolvingin methanol and precipitation using acetone. The product was thendissolved in DW and dialysed extensively against DW.

Example 9 Preparation of Amino-HPMA

HPMA (4.0 g) was dissolved in DMSO (100 ml). A 1.5 ml aliquot of DIEAwas added followed by 1.26 gm of solid CDI (1,1′-carbonyldiimidazole).The HPMA was activated for 45 min, whereupon an excess of1,6-diaminohexane (4.0 g) was added. The reaction proceeded for 2 h, atwhich time the product was dialysed to remove unreacted amines. Thefinal product was lyophilized.

Example 10 Preparation of Lysyl-HPMA

HPMA polymer (100K<MW<300K, 2.8 g) was dissolved in DMF (40 mL). DIEA(560 μL) was added, followed by Disuccinimidyl carbonate (1512 mg) andthe mixture stirred at room temperature under N₂ overnight. Lysine wasdissolved at 100 mg/ml in 10% sodium carbonate. 1 gm lysine was added tothe derivatized-HPMA and allowed to react overnight. The product waspurified by dialysis to remove free DSC and lysine.

Example 11 Preparation of Methotrexate-HPMA Polymers Targeted withBiotin

HPMA polymer (100K<MW<300K, 2.8 g) was dissolved in DMF (40 mL). DIEA(560 μL) was added, followed by Disuccinimidyl carbonate (1512 mg) andthe mixture stirred at room temperature under N₂ overnight.Methotrexate-Gly-Phe-Leu-Gly-Lysine (630 mg) was added and the mixturestirred for 30 min.

Biotin-Lys (MW 372, 80 mg dissolved in 1% NaHCO₃ solution) was added andthe mixture was reacted overnight. The Polymer-product was precipitatedby the addition of ethyl acetate and the pellet collected bycentrifugation at 5000 rpm. The pellet was washed twice with acetone,and the resultant product was dissolved in DW and dialysed extensivelyagainst ammonium hydrogen carbonate solution. The product waslyophilysed.

Example 12 Preparation of Methotrexate-Dextrin Polymers Targeted withBiotin

Dextrin polymer (100K<MW<300K, 2.8 g) was dissolved in DMF (40 mL). DIEA(560 μL) was added, followed by Disuccinimidyl carbonate (1512 mg) andthe mixture stirred at room temperature under N₂ overnight.Methotrexate-Gly-Phe-Leu-Gly-Lysine (630 mg) was added and the mixturestirred for 30 min.

Biotin-Lys (MW 372, 80 mg dissolved in 1% NaHCO₃ solution) was added andthe mixture was reacted overnight. The Polymer-product was precipitatedby the addition of ethyl acetate and the pellet collected bycentrifugation at 5000 rpm. The pellet was washed twice with acetone,and the resultant product was dissolved in DW and dialysed extensivelyagainst ammonium hydrogen carbonate solution.

The product was lyophilysed.

Example 13 Preparation of Aminohexyl-carboxymethyl Cellulose (CMC)

CMC (low viscosity) was dissolved at 25 mg/ml in DW (2 gm/40 ml). NHS(150 mg dissolved @ 100 mg/ml in acetone) was added followed by 300 mgdry EDAC. The CMC was reacted for 15 minutes, whereupon 5 ml 1 Mdiaminohexane pH 9.5 was added and allowed to react O/WE. The productwas dialysed exhaustively against DW. The product was then filtersterilized.

Example 14 Biotin Derivatisation of Polymers

Biotin (90 mg) was dissolved in DMSO (5.0 ml). DIEA (75 μL) was added,followed by TSTU((O-(N-Succinimidyl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate) (180 mg). The biotin was activated for 10 min, then1.0 g Polymer (amino-HPMA, or amino-hexyl-CMC) dissolved in DMSO (50 ml)was added to the activated biotin solution and reacted overnight. Theproduct was dialysed extensively to ensure removal of unreacted acid.The product was lyophilized.

Example 15 Preparation of Methotrexate-GFLG-HPMA-Biotin

Methotrexate-GFLG-OH (FW 828, 36 mg, 3 ×biotin) was dissolved in DMSO (5ml). DIEA (20 μL) was added, followed by TSTU (35 mg). The methotrexatewas activated for 10 min. The polymer (100 mg) (Aminohexyl-HPMA orbiotin-hexyl-HPMA) dissolved in DMSO (15 ml) was added to the activatedDrug-GFLG-acid solution and reacted 60 min. The product was dialysedextensively to ensure removal of unreacted acid and lyophilysed.

Example 16 Preparation of Methotrexate-GFLG-CMC-Biotin

Methotrexate-GFLG-OH (FW 828, 36 mg, 3×biotin) was dissolved in DMSO (5ml). DIEA (20 μL) was added, followed by TSTU (35 mg). The methotrexatewas activated for 10 min. The polymer (100 mg) (Aminohexyl-CMC orbiotin-hexyl-CMC) dissolved in DMSO (15 ml) was added to the activatedDrug-GFLG-acid solution and reacted 60 min. The product was dialysedextensively to ensure removal of unreacted acid and lyophilysed.

Example 15 Preparation of Clorambucil-GFLG-HPMA-Biotin

Chlorambucil-GFLG-OH (FW 678, 29 mg, 3×biotin) was dissolved in DMSO (5ml). DIEA (20 μL) was added, followed by TSTU (35 mg). The chlorambucilwas activated for 10 min. The polymer (100 mg) (Aminohexyl-HPMA orbiotin-hexyl-HPMA) dissolved in DMSO (15 ml) was added to the activatedDrug-GFLG-acid solution and reacted 60 min. The product was dialysedextensively to ensure removal of unreacted acid and lyophilysed.

Example 16 Preparation of Chlorambucil-GFLG-CMC-Biotin

Chlorambucil-GFLG-OH (FW 678, 29 mg, 3×biotin) was dissolved in DMSO (5ml). DIEA (20 μL) was added, followed by TSTU (35 mg). The chlorambucilwas activated for 10 min. The polymer (100 mg) (Aminohexyl-CMC orbiotin-hexyl-CMC) dissolved in DMSO (15 ml) was added to the activatedDrug-GFLG-acid solution and reacted 60 min. The product was dialysedextensively to ensure removal of unreacted acid and lyophilysed.

Example 17 Preparation of HPMA-hexylaminosuccinate

Aminohexyl-HPMA (300 mg) was dissolved in DMSO (5 ml) and succinicanhydride (100 mg) and DIEA (100 μL) added. The polymer was reactedovernight then dialysed extensively against DW and lyophilysed.

Example 18 Preparation of Daunomycin-GLFG-HPMA-Biotin

HPMA-hexylaminosuccinic acid (35 mg) was dissolved in DMSO (2.0 ml).TSTU (18 mg) was added and activated for 10 min. H₂N-GFLG-Daunomycin (FW938, 3×biotin, 4.4 mg) was added and allowed to react for 5 min. Fortargeted polymers 6-aminohexyl-biotin (3 mg, designed to give 20%loading) was added and reacted for 1 h. The product was dialysed toremove unconjugated reagents. The final product was concentrated usingan AMICON positive pressure stirred cell with 10K membrane.

Example 19 Preparation of MTX-GFLG-MLP-Biotin

MTX-GFLG-OH (FW 828, 25 mg) was dissolved in DMSO (2 ml). TEA (5 μl) wasadded, followed by TSTU (15 mg, 1.2 equiv.). The reaction was allowed toproceed for 10 min, afterwhich 13 mg MLP Polymer dissolved in DMSO (0.5ml) was added and reacted for 60 min. For preparation of targetedpolymers biotin (8 mg) dissolved in DMSO (0.8 ml) was activated withTSTU (8.5 mg) for 10 min and then the activated targeting agent wasadded to MTX-GFLG-MLP mixture. The reaction proceeded for 60 min. 0.1 MTris pH 7.5(5 ml) was added and stirred 1 h. The product was dialysedextensively and lyophilysed.

Example 20 Demonstration of Biotin-Mediated Targeting of Polymers

In order to examine the potential utility of biotin as a targeting agentfor polymer-drug conjugates, Lysyl-HPMA was substituted with rhodamineusing rhodamine-isothiocyanate using standard methods. An aliquot of theRho-HPMA was then further reacted with biotin, to produce abiotin-substituted-Rhodamine-HPMA. Control polymers were preparedwithout biotin. For tumour accumulation studies, various strains of micebearing a variety of tumours were injected intraperitoneally with 5mg/kg Rhodamine conjugated to the HPMA polymers. Six hours afterinjection, the mice were sacrificed, their tumours removed andcryo-embedded before cryostatic sectioning. The level of accumulation ofthe Rhodamine-HPMA was determined by fluorescent microscopy using aZeiss microscope equiped with Axioplan software. Representative sectionsare shown in FIG. 1. The data shows that the level of polymer uptake byP815 tumour cells can be enhanced by biotin derivatization of therhodamine labelled polymers, as indicated by red staining. Blue stainingis BisBenzamide staining of cell nuclei.

Example 21 Increased Localization of Targeted HPMA in L1210FR TumourCells in DBA/2 Mice with Biotin

Preliminary experiments were performed in L1210FR mice to determinewhether Rhodamine-labelled polymers would localize to ascites cells inL1210 FR tumours injected IP.

Lysyl-HPMA was derivatized with Fluorescein (using FITC) or rhodamine(using TRITC) using standard methods. Derivatization was aimed at 5%substitution, however, with FITC this was too substituted and resultedin an insoluble polymer, therefore substitution was backed off to 2.5%.For the production of targeted polymers the Glycyl-5′O-VB₁₂, and folatewere activated with TSTU and used to substitute the remaining aminogroups on the fluorescent polymers. Polymers were also biotinylated withNHS-biotin. Free reagents were removed by dialysis.

Mice were injected IP with 100 ug polymer and left for 5 hours, at whichtime the mice euthanased by cervical dislocation. The peritioneal cavitywas then flushed with 5 ml of 3.8% trisodium citrate, and ascites fluid,containing cells, was then aspirated from the peritioneal cavity. Thefluid was kept at 4° C. ON before processing. The quantity of cells inthe peritoneal wash out was determined by centrifuging the fluid andmeasuring the volume of the pellet. A fixed quantity of cells were thendiluted out two-fold in an ELISA plate for measurement of fluorescenceand determination of the level of uptake of fluorescent polymer.

Cells were also placed on slides for microscopic examination ofinternalized fluorescence. Fluorescence determination was performed on aZeiss Axioplan fluorescent microscope, and photographed. Results areshown in FIGS. 2. and 3. Examination of the amount of fluorescentpolymer taken up by isolated ascites cells taken from mice at the timeof sacrifice showed greatly increased uptake of all targeted polymers.Greatest uptake was seen with the biotinylated polymers, followed byfolate and vitamin B12 as targeting agents.

Example 22 Preparation of Nanospheres

Nanospheres can be formed by a number of techniques common to thoseknowledgeable in the art, including: Solvent evaporation, Complexcoacervation, Polymer/polymer incompatibility, Gelation, Interfacialpolymerization and Thermal denaturation.

An effective amount of the complex is formulated with a pharmaceuticallyacceptable carrier, diluent or excipient to provide a medicament foradministration to a patient requiring treatment of the conditionsoutlined in the body of the specification. The formulation is preparedusing standard pharmaceutical techniques.

It is recognized that a number of factors will affect the determinationof an appropriate dosage for a particular host. Such factors include theage, weight, sex, general health and concurrent disease states of thehost. The determination of the appropriate dose level for the particularhost is performed by standard pharmaceutical techniques.

Example 23 Preparation of Nanospheres by Coacervation

Almost any protein can be used as the matrix for entrapping drug via thedesolvation technique, however preferred proteins according to theinvention include bovine serum albumen (BSA), Ovalbumen (OA) andcollagen.

BSA Nanospheres Formed by Desolvation.

Nanospheres were prepared by coacervation of BSA following desolvation,according to the method of Oppenheim (Oppenheim, 1984, Oppenheim et al1984, 1982), Briefly a 40% ammonium sulphate solution was added dropwiseto a solution of 1% BSA containing 0.5% Tween 20 and the turbiditymonitored by Klett readings, until the turbidity rose rapidly. At thispoint (determined by experimentation) the solution was placed in anultra-turrax and 600 ul of glutaraldehyde added to cross-link thenanoparticles. Cross-linking was stopped by the addition of a solutionof 12% sodium metabisulfite. Particles were then washed extensively withdistilled water prior to coupling to the NHS-derivative of biotin

Example 24 Incorporation of 5-fluorouracil

The antimitotic, 5-fluorouracil, was dissolved at 10 g/100 ml of theBSA/Tween solution. Desolvation and cross-linking was carried out asdescribed in Example 23.

Example 25 Coupling of Biotin to Nanospheres

Proteinaceous nanospheres (prepared by the method described in Example23) were surface coated with biotin by reaction of biotin with EDAC andNHS followed by addition to the preformed nanospheres.

Example 26 Preparation of Biotin-Lipid Complexes for HydrophobicInsertion into Nanospheres

In order to link biotin to the surface of nanospheres which have noreadily available chemical groups suitable for chemical conjugation, itis possible to prepare a complex of biotin to an hydrophobic moietywhich can insert, non-covalently, into the surface of the nanospheres.Such a molecule is easily added at the time of formation of thenanospheres. The strength of the hydrophobic association is such thatthere is only a very slow dissociation of the biotin from thenanospheres under physiological conditions.

a) Preparation of biotin-phosphatidyl ethanolamine (biotin-PEA)

Phosphatidylethanolamine (100 mg) was dissolved in 2 mlchloroform/methanol (50:50, v/v). Biotin (100 mg) was added to themixture. The biotin was then cross-linked to the PEA by the addition of200 mg of the carbodiimide,1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide (EDC or EDAC). Thereaction was allowed to proceed for 90 minutes prior to the addition ofthe biotin-PEA to nanospheres.

b) Preparation of other complexes between biotin and an hydrophobicmoiety.

Covalent complexes can be made between analogues of biotin and almostany aliphatic or aromatic chains or amphipathic containing a watersoluble head group suitable for conjugation and a lipid soluble tailsuitable for hydrophobic association within an hydrophobic environment.Thus, any lipid (saturated, unsaturated or polyunsaturated) which has acarboxylic acid head group, such as Oleic acid, octanoic acid, linoleicacid or glycerophophoric acids may be directly conjugated to anamino-biotin derivative using a suitable carbodiimide (EDAC or DCC, forexample). Similarly any amphiphathic molecule possessing an amino-group(amino-hexane, amino-decane, amino-dodecane, phosphatidyl-ethanolamine,may be conjugated directly to carboxy-biotin using carbodiimides.

Example 27 Preparation of Biotin-Nanospheres by Solvent Evaporation

a) Preparation of biotin-PEA-[Polymethylmethacrylate] nanospheres

Polymethylmethacrylate (PMM, Polysciences)(MW 12,000; 500 mg) wasdissolved in 2 ml of dichloromethane (DCM). The PMM in DCM was thenadded dropwise to 20 ml of 0.25% Polyvinylalcohol (PVA) whilehomogenizing at 13,500 rpm with a Janke & Kunkel Ultraturrax. After 1minute, 200 ul of biotin-PEA was added and stirred gently overnight. Thenanospheres were then harvested by centrifugation, washed three timeswith water and lyophilized.

b) Preparation of biotin-[PEA-Poly-lactic acid] nanospheres.

Poly-lactic acid (PLA, Polysciences)(MW 50,000; 500 mg) was dissolved in3 ml of DCM and then homogenized into 20 1% PVA at 13,500 rpm onUltraturrax T25 with an S25F probe for 5 minutes. biotin-PEA (400 ul)was added while the solution was stirred gently. Nanospheres wereharvested as described above.

c) Preparation of biotin-PEA-[Poly-Hydroxy-butyrate/valerate]nanospheres

Poly-Hydroxy-butyrate/valerate (9% valerate) (ICI; 500 mg) was dissolvedin 5 ml of DCM and homogenized into 20 ml 1% PVA at 13,500 rpm onUltraturrax T25 with an S25F probe for 5 minutes. biotin-PEA (400 ul)was added and the spheres processed as described in 8b.

Example 28 Covalent Conjugation of Biotin to Nanospheres with SurfaceCarboxyl Groups

A general method for the conjugation of biotin to the surface ofnanospheres made from polymers with free carboxyl groups is outlinedbelow. The specific example utilizes commercially availablecarboxyl-modified nanospheres.

Polysciences Fluoresbrite™ carboxylate Nanospheres (2.5% Solids Latex)were obtained from Polysciences in sizes of 0.045 um, 0.49 um, 2.2 umand 9.97 um. One ml of each of the preparations was washed extensivelywith DW and resuspended in 200 ul of distilled water. To eachpreparation was added 1.5 mg aminohexyl biotin then 5 mg of EDAC. Eachpreparation was allowed to react overnight, after which unreactedmaterial was removed by repeated washing with DW or by dialysis againstDW.

Example 29 Surface Derivatization of Nanospheres

Many polymers used in the preparation of nanospheres by solventevaporation do not contain functional groups for direct conjugation tobiotin or its functionalized analogues, however it is possible to modifythe surface of the preformed nanospheres to introduce functional groupssuitable for conjugation to biotin.

a) Surface derivatization of Polylactic acid (PLA) nanospheres

Preformed PLA nanospheres (10 mg) were gently suspended in distilledwater (DW; 350 ul) by rotation on a rotary shaker for 2 hours. Hydrazinehydrate (10 ul) was added and the suspension was shaken overnight atroom temperature. The spheres were spun down and repeatedly washed withwater by re-suspension and centrifugation. The washing procedure wasrepeated until the supernatant failed to give a positive hydrazine test(purple colour upon reaction with a solution of TNBS; 1 mg /ml). Thespheres were washed a further two times and the wet pellet used directlyfor conjugation to biotin.

b) Conjugation of biotin to hydrazine modified PLA nanospheres

A sample of the hydrazine modified PLA nanospheres (3 ul wet pellet) wassuspended in DW (250 ul). Aqueous solutions of biotin (10 mg/ml, 400 ul)and EDAC (100 mg/ml, 100 ul) were added and the reaction mixture shakenovernight at room temperature. The suspension was spun down and thesupernatant removed. The pellet was washed repeatedly with DW (6washes). The residual pellet, was vacuum dried.

Two control reactions were performed concurrently with the aboveconjugation. In the first a 3 mg sample of hydrazine-modified PLAnanospheres was treated with the biotin as described above but DW wasused in place of the EDAC solution. In the second control a 2 mg sampleof unmodified PLA nanospheres was treated with both biotin and EDAC asdescribed above. For both controls the pellet remaining after repeatedwashing was a clear white colour with no evidence of any associatedbiotin.

Example 30 Preparation of Isobutyl-Cyanoacrylate Nanocapsules,Surface-Coated with Biotin

Nanocapsules suitable for biodistribution studies were prepared with¹²⁵I-insulin as an internal marker. Briefly, 10 mg insulin was dissolvedat 10 mg/ml in 0.1 M HCl. An aliquot (1 μl) of ¹²⁵I-insulin was added tothe cold insulin, which was mixed with 100 μl Miglyol™ and vortexed.EtOH (10 ml) was added to the insulin/Miglyol™ mix and mixed byvortexing. IBCA (100 μl, Sicomet) was added to the clear solution, whichwas immediately added to 60 ml 0.25% F-127. After 30 minutes thepreparation was split into 2 equal halves. One half was left to stirovernight, whilst to the other half was added 27 mgbiotin-PEG-octadecanoic acid (80 mg/ml in EtOH). The solution was leftto stir overnight. Both solutions were then treated in a similarfashion. Large aggregates were removed by centrifugation at 10K for 20minutes. Both particle preparations were concentrated and washed in aAmicon positive pressure filtration unit using a 300,000 MW cut offmembrane. Particles were stabilized by surface cross-linking withdi-succinimidyl-2-aminoethyl-2-amino-2-benzyl-ethanoate (DSAB). DSAB wasconverted to the NHS-ester as follows. DSAB (40 mg) was dissolved in anequal weight of DMF, to which was added NHS (24 mg, 240 μl DMF). DCC(Dicyclohexylcarbodiimide, 44 mg, 440 μl, made up fresh) was then addedto the DSAB mixture and allowed to activate for 20′ while stirringrapidly. The DSAB-NHS-ester was added at 0.32 mg per 2.1 mgnanocapsules, and left to stir O/N. The particles were then dialysedbefore use in biodistribution studies.

Example 31 Identification of Cells that Over-Express Receptors Involvedin Vitamin Uptake

Cells from various tumour cell lines were allowed to grow for 2 days onglass slides incubated in appropriate media at 37° C. in 5% CO₂. After 2days the media was removed and was replaced with spent culture mediacontaining a Rhodamine-HPMA polymer to which was bound vitamin B12,folate or biotin. Cells were incubated for a further 5 hours at 37° C.in 5% CO₂. At this time the media was removed and uptake into the cellswas assessed by fluorescent microscopy of internalization of therhodamine fluorophore. Uptake was determined on a relative scale. TABLE1 Over-expression of vitamin receptors amongst various Tumour Cell linesTumour Polymer Folate VB12 Biotin O157 Balb/C Bcell Lymph +/− +/− +/−+/− BW5147 AKR/J Lymphoma +/− +/− +/− +/− B16 C57/BI Melanoma − − − −LL-2 C57/BI Lung − − − − HCT-116 Balb/C-Nu Colon Carcin + − − − L1210DBA/2 Leukemia − +/− +/− − L1210FR DBA/2 Leukemia − ++ + +++ Ov 2008Balb/C-Nu Ovarian − +++ − ++ ID8 C57/BI Ovarian − +++ − ++ Ovcar Ovarian− +++ − ++ Colo-26 Balb/C Colon Carcin − +/− ++ +++ P815 DBA/2Mastocytoma − +/− ++ +++ M109 Balb/C Lung − + +++ +++ RENCA Balb/C Renalcell − + +++ +++ 4T1 Balb/C Breast − + +++ +++ JC Balb/C Breast − + ++++++ MMT060562 Balb/C Breast − + +++ +++

As can be seen from Table 1, all tumours that over-expressed receptorsinvolved in Vitamin B12, or folate uptake, also over-expressed receptorsinvolved in biotin binding. Representative FIGS. 4 to 9 show uptake intoOv2008, RENCA, 4T1, JC and MMTO60562 are also attached.

Example 32 Enhanced Killing of Colo-26 Tumour Cells Treated withDox-GFLG-HPMA Targeted with Biotin

Dox was covalently linked to C-terminus of the tetrapeptideNH₂Gly-Phe-Leu-Gly-COOH as described above. The tetrapeptide-Doxconjugate was then linked to the HPMA polymer, after which the polymerwas modified with the targeting agents biotin, folate and vitamin B₁₂.Non-conjugated material was removed by extensive dialysis. The Colo-26tumour (2×10⁶ cells) was injected into Balb/C mice and allowed to growfor 7 days, at which time a small lump was apparent at the site ofsubcutaneous injection of the tumour. Mice were then injectedintravenously with a dose of 20 mg/kg doxorubicin, either alone orconjugated to the polymer, on each of 3 successive days. The tumour wasthen allowed to grow in the mice, and its size determined via a two waymeasuremtn using Venier calipers. Data is presented as the averagetumour weight of the mice over time.

As can be seen from FIG. 10, substantial reduction in tumour mass wasseen in the group that received the biotin-targeted Dox-TP-HPMA. Thebiotin dependency of this increased killing was shown by the reducedefficacy of this polymer conjugate in the presence of excess biotin. Infact the biotin-targeted polymer group was the only group that showedenhanced killing over and above that seen with the polymer alone.

Example 33 Preparation of DNM-HPMA Hydrazone

Oxidised-PHPMA (3000 mg) was dissolved in MeOH (30 mL). Hydrazidyl-DNM(300 mg) was added to each aliquot, followed by 1 Drop AcOH. The mixturewas stirred for 3 h, after which, Hydrazidyl biotin (95 mg) was addedand the mixture was then stirred overnight. The product thenprecipitated upon addition of ethyl acetate and was isolated bycentrifugation. The pellet was washed with acetone and again isolated bycentrifugation. The pellet was dissolved in PBS and dialysed at pH 7.4.

Example 34 Preparation of Biotin-poly(HPMA)-GGG-Ame

Poly(HPMA)-GGG-Ame (7.5 g) was dissolved in DMSO (75 mL) and TEA (400μL) was added. DSC (disuccinimidyl carbonate, 405 mg) was then added andthe mixture stirred for 24 h at room temperature (22° C.). AE-Biotin (MW286, 260 mg) was added and the mixture stirred for a further 1 hour.Ethyl acetate (4 volumes) was added to precipitate the polymer and themixture spun at 5000 rpm for 10 min and the supernatant removed.Acetonitrile (4 volumes) was added to resuspend the polymer, after whichthe mixture was spun at 5000 rpm for 10 min and the supernatant removed.The pellet was dissolved in distilled water and purified by tangentialflow filtration, at which time the mixture was lyophilysed fromwater/MeCN.

Example 35 Preparation of Biotin-poly(HPMA)-GG-Ame

PHPMA-GG-ONp (KBT196-200A, 100 mg) was dissolved in DMSO (1 mL) andAE-Biotin (3.0 mg) added. The mixture was stirred for 1 h beforeaddition of Hydrazine.2HCl (100 mg) dissolved in 3 mL MeOH containingTEA (0.5 mL). Targeted polymer was then added to the solution ofhydrazine, which was reacted for 2 h. The resultant product was dilutedwith DW and dialysed extensively against ammonium hydrogen carbonatesolution and then DW. Product was lyophilysed.

Example 36 Conjugation of Dox to Biotin-poly(HPMA)-GG-Ame Hydrazone

The biotin-targeted PHPMA bearing hydrazidyl functionality was dissolvedin MeOH (1.0 mL) and DNM (10 mg) added plus 1 Drop AcOH. The mixture wasstirred for 2 days and the product then precipitated upon addition ofethyl acetate. The pellet was washed with acetone and again isolated bycentrifugation. The pellet was dissolved in PBS and dialysed at pH 7.4.

Example 37 Preparation of Dox-TP-Lysyl-poly(HPMA)

HPMA polymer (1.5 g) was dissolved in DMF (30 mL) and DIEA (250 μL)added. Disuccinimidyl carbonate (250 mg) was added and the mixturestirred at room temperature under N₂ overnight. Lys-Succ-GFLG-DNM (MW1174, 200 mg) was then added and the mixture stirred for 30 min.Biotin-Lys (MW 372, 50 mg) was added to the solution which was thenallowed to react overnight. The resultant product was diluted with DWand dialysed extensively against ammonium hydrogen carbonate solution.The product was lyophilysed.

Example 38 Preparation of HYNIC-HPMA Suitable for Use with ^(99m)Tc

HPMA-GFLG-en polymer (AT-119-134, 4.0 g) was dissolved in DMSO (20 mL)and Boc-HYNIC-OSu (Succinimidyl 6-BOC-hydrazinonicotinate, MW 350, 300mg) added. The mixture was stirred for 1 h. Separately, biotin (400 mg)was dissolved in 6.0 ML DMSO, DIEA (240 μL) was added, followed by TSTU(520 mg) and the mixture activated for 15 min. The activated vitamin wasadded to the HPMA-GFLG-en-HYNIC-Boc prepared above, and stirred for 2 h.Free amino groups were blocked by the addition of a solution of aceticanhydride (60 μL) in DMSO (500 μL) containing NHS (70 mg). The mixturewas stirred for 2 h. The product was precipitated by addition of ethylacetate, and isolated by centrifugation at 5000 rpm for 10 min. Thepellet was washed by sonication in MeCN and again isolated bycentrifugation. This pellet was then dissolved in TFA (20.0 mL) andafter 20 min the product precipitated on addition of petroleumether/ethyl acetate (100 mL). The pellet was washed by sonication inMeCN/ethyl acetate/light petroleum and again isolated by centrifugation.The pellet was then washed with acetone and spun at 5000 rpm for 5 min.The resultant pellet was redissolved in carbonate buffer and the polymerwas dialysed extensively using MWCO 3500. The product was thenlyophilysed.

Example 39 Preparation of Mtx-poly(HPMA)

HPMA polymer (8.0 g) was dissolved in DMSO (120 mL), to which DIEA (2000μL) and Disuccinimidyl carbonate (2000 mg) were added sequentially, andthe mixture stirred at room temperature under N₂ overnight. MTX-GFLG-Lys(1600 mg, α, γ mixture) was added and the mixture stirred for 30 min.The reaction mixture was divided into 4 aliquots. AH-VB₁₂ (MW 1497, 360mg), FA-Lys (MW 569, 137 mg) and Biotin-Lys (MW 372, 89 mg) were addedto separate aliquots. Water was added to aid solubility. The mixtureswere reacted overnight. The resultant product was dissolved in DW anddialysed extensively against ammonium hydrogen carbonate solution thenDW. The product was lyophilysed.

Example 40 Preparation of Mtx-GFLG-Lys poly(HPMA)

MTX-(OMe)-GFLG-OH (FW 842, 180 mg) dissolved in DMSO (4 mL), to whichwas added DIEA (30 μL) followed by HPPyU (95 mg). The material wasactivated for 15 min prior to addition to 900 mg Polymer (Lys-HPMA)dissolved in DMSO (20 mL). The reaction proceeded for 60 min, at whichit was divided into 4 aliquots in preparation for addition of targetingagents.

Example 41 Preparation of VB12/folate/biotin-[Mtx-GFLG-Lys poly(HPMA)]

Separate aliquots of VB₁₂-Gly acid (100 mg) or FA (30 mg) or Biotin (MW244, 17 mg) were dissolved in DMSO (1.0 mL), to which was added DIEA (10μL) and pyBOP (43 mg). The solution was activated for 25 min, beforeaddition of the activated acids to the Mtx-GFLG-Lys poly(HPMA) polymeraliquot. The reaction proceeded for 2 h before the addition of 0.1 MNaOH solution to pH 11 to remove methyl ester. Deprotection proceededfor 20 min, at which time it was dialysed extensively and thenlyophilysed.

Example 42 Preparation of Poly(HPMA)-GFLG-en-Biotin

Biotin (MW 244, 250 mg) was dissolved in DMSO (3 mL) and TEA (150 μL)was added prior to addition of TSTU (MW 301, 308 mg) and activation for15 min. Separately, PHPMA-GFLG-en (22 kDa, AT-119-134, 1.0 g) wasdissolved in DMSO (7 mL). The activated biotin was added to the rapidlystirring PHPMA-GFLG-en solution and the reaction was stirred for 4 h.The product was diluted with distilled water and dialysed extensivelyagainst DW (MWCO 3400). The dialysed solution was lyophilysed to affordthe biotinylated polymer as a slightly brown powder.

Example 43 Preparation of Biotin-Targeted (poly(HPMA)-GGG-Ama-Pt-DACH

An aliquot of (poly(HPMA)-GGG-Ama-Pt-DACH, 200 mg) was dissolved inDMF/MeOH (1:1, 2 mL) and DIEA (10 μL) was added, before addition ofpyBOP (MW 520, 12.5 mg), with stirring. AE-Desthiobiotin (5.7 mg) wasadded to the aliquot and the mixture stirred for 2 h beforeprecipitation of the product by addition of ethyl acetate.

After centrifugation the pellet was sonicated in acetone and isolatedagain by centrifugation. The product was dissolved in DW and purifiedusing Centricon 20's (5 kDa membrane) spinning at 4000 rpm for 30 min.The pellet was washed 3 more times. The product was lyophilysed.

Example 44 Preparation of poly(HPMA)-GFLG-en Succ-DNM

HPMA-GFLG-AE (HPMA-GFLG-en, AT-119-64, 600 mg) was dissolved in DMSO (5mL). Succinyl-DNM (MW 627, 100 mg) was dissolved separately in DMSO (1mL) and DIEA (20 μL) was added. HPPyU (70 mg) was added and the acid wasactivated for 15 min. The activated acid was added to HPMA-GFLG-ensolution and reacted for 1 h. The mixture was divided into 3×2 mLaliquots for subsequent targeting.

Example 45 Preparation of VB12/FA-en-GLFG-HPMA-GFLG-en-Succ-DNM

Either VB₁₂-Gly-OH (MW 1456, 75 mg) or FA (MW 441, 22 mg) was dissolvedin DMSO (500 μL) and DIEA (9 μL) was added. HPPyU (22 mg) was added andthe acid was activated for 15 min. Activated acid was added toHPMA-GFLG-en-Succ-DNM solution and reacted for 2 h. The product wasdiluted with water and dialysed extensively

Example 46 Preparation of HPMA-GFLG-en-Mtx

HPMA-GFLG-AE (HPMA-GFLG-en, AT-119-64, 3000 mg) was dissolved in DMSO(40 mL). MTX (MW 454, 250 mg) was dissolved separately in DMSO (5 mL)and DIEA (200 μl) was added. pyBOP (340 mg) was added and the acid wasactivated for 55 min. Activated acid was added to HPMA-GFLG-en solutionand reacted for 1 h. The mixture was divided into 4 aliquots forsubsequent targeting.

Example 47 Preparation of VB_(12/)FA/Biotin-en-HPMA-GFLG-en-Mtx

Either VB₁₂-Gly-OH (MW 1456, 125 mg) or FA (MW 441, 40 mg) or Biotin (MW244, 21 mg) was dissolved in DMSO (1500 μL) and DIEA (20 μL) was added.TSTU (31 mg) was added and the acid was activated for 15 min. Activatedacid was added to an aliquot of HPMA-GFLG-en-MTX and reacted for 2 h.The product was precipitated with ethyl acetate and collected bycentrifugation. The pellet was dissolved in water and dialysedextensively. Product was dialysed.

Example 48 Preparation of Mtx-HSA

Mtx was dissolved at 100 mg/ml in DMSO (88 mg). PyBOP (100 mg/ml inDMSO, 114 mg) plus 176 μl DIEA was added to the Mtx, and allowed toreact for 60 minutes. HSA was dissolved at 100 mg/ml in 1% NaHCO₃ (880mg), and the activated Mtx added to it and allowed to react overnight.The free Mtx was separated from Mtx-BSA on Sephacryl S-200 in PBS,before dialysis and lyophilization of the product.

Example 48 Preparation of Biotin-Modified Mtx-HSA

Biotin was dissolved at 100 mg/ml in DMSO. TSTU, dissolved at 130 mg/mlin DMSO, was added to the biotin as well as 100 μl TEA. The biotin wasactivated for 30 mins, before addition to Mtx-HSA (100 mg/ml in 1%sodium bicarbonate).

Example 49 Preparation of Dox-DSP-HSA

Doxorubicin (Dox) was dissolved at 100 mg/ml in DMF. A 4-molar excess ofDSP was added to the Dox and allowed to react for 30 minutes. Theproduct was precipitated with acetonitrile to 80%, resuspended in DMFand added at 5% w/w to HSA dissolved at 100 mg/ml in 1% NaHCO3. Thematerial was allowed to react O/N, and was purified by dialysis. Theproduct was biotinylated as described previously.

INDUSTRIAL APPLICATIONS

The present invention provides a simple and novel technique for thespecific targeting of pharmaceuticals to tumour cells using polymers.This technique has commercial applications in enhancing the efficacy ofcurrent tumour treatments as well as potential applications in treatmentof inflammatory conditions.

The invention has been described herein, with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. However, a person havingordinary skill in the art will readily recognise that many of thecomponents and parameters may be varied or modified to a certain extentwithout departing from the scope of the invention. Furthermore, titles,headings, or the like are provided to enhance the reader's comprehensionof this document, and should not be read as limiting the scope of thepresent invention.

The entire disclosures of all applications, patents and publications,cited herein, if any, are hereby incorporated by reference.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually of collectively, andany and all combinations of any two or more of said steps or features.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in the field ofendeavour.

The following U.S. Patents, foreign patents and applications and otherreferences are incorporated herein by reference. U.S. Patents and PatentApplications Low et al 5,416,016 1995 Russell-Jones et al 5,428,023 1995Russell-Jones et al 5,449,720 1995 Russell-Jones et al 5,548,064 1996Russell-Jones et al 5,589,463 1996 Russell-Jones et al 5,807,832 1998Russell-Jones et al 5,869,466 1999 Russell-Jones et al 6,150,341 2000Russell-Jones et al 6,159,502 2000 Russell-Jones et al 6,262,253 2001Grissom, C. B, et al. 6,315,978 2001 Foreign Patents and ApplicationsMcEwan et al PCT WO0066091 2000 Grissom, C. B. et al U.S. Pat Appl 200220020049154

Selected References

Oppenheim R. C. (1984) in “Polymeric Microparticles” (Guiot, P andCouvreur, P. Eds.) CRC Press, Boca Raton.

Oppenheim R. C., Gipps, E. M. Forbes, J. F. and Whitehead R. H. (1984)in “Nanospheres and Drug Therapy” (Davis, S. S., Illum, L., McVie, J. G.and Tomlinson, E. Eds) Elsevier Science Publishers B.V.

Oppenheim, R. C., Stewart, N. F., Gordon, L. and Patel, H. M. (1982)Drug Devel. Indust. Pharm. 8:531-546.

Allen, R. H. and Majerus, P. W. (1972) J. Biol. Chem. 247:7702-7717.

Yamada, R.-H and H. P. C. Hogenkamp. (1972) J. Biol.Chem. 247:6266-6270.

1. A macromolecular conjugate comprising a support to which is coupledat least one targeting molecule in association with an active substance,wherein said targeting molecule is biotin or an analogue thereofpossessing binding activity to a biotin receptor.
 2. A conjugate ofclaim 1, wherein the support is a polymer.
 3. A conjugate of claim 1,wherein the support is a nanoparticle.
 4. A conjugate of claim 1 havingthe general formula:(B−Q)_(n)−P−(Q′−A)_(m) wherein B is biotin or a derivative thereof whichis a carrier that binds to a biotin receptor n, the molar substitutionratio of B in the conjugate, is a number from 1.0 to about 50; P is apharmaceutically acceptable linear, branched or dendritic polymer; A isa pharmaceutically or diagnostic active substance; m is a number greaterthan 1.0 to about 1000; and Q and Q′ are independently a covalent bond,or a spacer compound linking biotin, P and A by covalent bonds.
 5. Theconjugate according to claim 4, wherein at least one of Q and Q′ is aspacer compound which contains a biodegradable portion.
 6. The conjugateaccording to claim 5, wherein said biodegradable portion is selectedfrom a disulfide bond, ester linkage, a γ-glutamyl-ε-lysine linkage anda diazo bond, and Gly-Phe-Leu-Gly.
 7. The conjugate according to claim4, wherein n is from 1.0 to about 1.5 and m is from 2 to about 200, morepreferably from about 10 to
 100. 8. A conjugate according to claim 4,wherein P is a biodegradable polymer.
 9. A conjuguate according to claim8, wherein said biodegradable polymer is selected from a biodegradablecarbohydrate polymer or a polymer of amino acids.
 10. A conjugateaccording to claim 4, wherein P is a non-biodegradable polymer.
 11. Aconjugate according to claim 10, wherein said non-biodegradable polymercomprises biodegradable side chains for covalent linkage to an activesubstance.
 12. A conjugate according to claim 4, wherein said polymer isselected from poly[N-(2-hydroxypropyl)-methacrylamide], dextran ordextran derivatives, chondroitan sulfate, water soluble polyurethanesformed by covalent linkage of PEG with lysine, poly(glutamic acid),poly(hydroxypropyl glutamine), branched chain polypeptides,carboxymethyl cellulose, dendrimers and PEG-dendrimers.
 13. A polymeraccording to claim 12, wherein said polymer is a branched chainpolypeptide optionally modified to provide multiple functional groupsfor coupling of an active substance.
 14. A conjugate according to claim5, wherein said spacer compound Q or Q′ has from 1 to 50 atoms in itsbackbone.
 15. A conjugate according to claim 4, wherein said spacer is adiradical spacer comprising optionally substituted alkylene C₁₋₅₀ moietyoptionally contained within the chain, double bonds, triple bonds, arylgroups and/or hetero atoms.
 16. A conjugate according to claim 15,wherein said spacer compound is derived from disuccinimidyl suberate(DSS), bis(sulfosuccinimidyl) suberate (BSS), ethyleneglycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (Sulfo-EGS), p-amino-phenylaceticacid, dithiobis(succinimidylpropionate) (DSP),3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidyltartarate (DST), disulfosuccinimidyl tartarate (Sulfo-DST),bis[2-(succinimidyloxycarbonyloxy)-ethylene]sulfone (BSOCOES),bis[2-(sulfosuccinimidooxycarbonyloxy)-ethylene]sulfone (Sulfo-BSOCOES),dimethyl adipimidate.2 HCl (DMA), dimethyl pimelimidate.2 HCl (DMP), ordimethyl suberimidate.2 HCl (DMS).
 17. A conjugate according to claim15, wherein said spacer compound is thiol cleavable.
 18. A conjugateaccording to claim 17, wherein said thiol-cleavable spacer is derivedfrom N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), iminothiolane,sulfosuccinimidyl 6-[3-(2-pyridyldithio) propionamido]hexanoate(Sulfo-LC-SPDP), succinimidyl 6-[3-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), sulfosuccinimidyl 6-[α-methyl-α-(2-pyridyldithio)toluamido] hexanoate (Sulfo-LC-SMPT),1,4-di[3′-(2′-pyridyldithio)propionamido]butane (DPDPB),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT) ordimethyl 3,3′dithiobispropionimidate.2 HCl (DTBP).
 19. A conjugateaccording to claim 1, wherein said active substance is a biologicallyactive toxin or a part thereof.
 20. A conjugate according to claim 19,wherein said toxin is selected from ricin, abrin, diphtheria toxin,modecin, tetanus toxin, mycotoxins, mellitin, α-amanitin, pokeweedantiviral protein and ribosome-inhibiting proteins, from wheat, barley,corn, rye, gelonin and maytansinoid.
 21. A conjugate according to claim1, wherein said active substance is an alkylating agent selected fromchlorambucil, cyclophosphamide, melphalan, cyclopropane; anthracyclineantitumor antibiotics such as doxorubicin, daunomycin, adriamycin,mitomycin C, [2-(hydroxymethyl)anthraquinone]; antimetabolites such asmethotrexate, dichloromethatrexate: cisplatin, carboplatin, andmetallopeptides containing platimun, copper, vanadium, iron, cobalt,gold, cadmium, zinc and nickel, DON, thymidine, pentamethylmelamin,dianhydrogalactitol, 5-Methyl-THF, anguidine, maytansine,neocarzinostatin, chlorozotocin, AZQ, 2′deoxycoformycin, PALA, AD-32,m-AMSA and misonidazole.
 22. A conjugate according to claim 1, whereinthe active substance is an imaging agent.
 23. A conjugate according toclaim 22, wherein the imaging agent is Rhodamine, fluorescein, Texasred, Acridine Orange, Alexa Fluor (various), Allophycocyanin,7-aminoactinomycin D, BOBO-1, BODIPY (various), Calcien, CalciumCrimson, Calcium green, Calcium Orange, 6-carboxyrhodamine 6G, Cascadeblue, Cascade yellow, DAPI DiA, DiD, Dil, DiO, DiR, ELF 97, Eosin, ERTracker Blue-White, EthD-1, Ethidium bromide, Fluo-3, Fluo-4, FM1-43,FM4-64, Fura-2, Fura Red, Hoechst 33258, Hoechst 33342,7-hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye, Lissaminerhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167, LysoSensorGreen, LysoSensor Yellow/Blu, Lysotracker Green FM, Magnesium Green,Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos,MitoTracker Red CMXRos, Monobromobimane, NBD amines, NeruoTrace 500/525green, Nile red, Oregon Green, Pacific Blue. POP-1, Propidium iodide,Rhodamine 110, Rhodamine Red, R-Phycoerythrin, Resorfin, RH414, Rhod-2,Rhodamine Green, Rhodamine 123, ROX dye, Sodium Green, SYTO blue(various), SYTO green (Various), SYTO orange (various), SYTOX blue,SYTOX green, SYTOX orange, Tetramethylrhodamine B, TOT-1, TOT-3,X-rhod-1, YOYO-1 or YOYO-3.
 24. A conjugate according to claim 1,wherein the active substance is a radionuclide.
 25. A conjugateaccording to claim 22 wherein the imaging agent is a radionuclide.
 26. Aconjugate according to claim 22 wherein the imaging agent is linked to apolymer.
 27. A conjugate according to claim 22 wherein the imaging agentis incorporated within and/or coated on a surface of a nanoparticle. 28.A conjugate according to claim 4 in which the pharmaceuticallyacceptable polymer has the sequence of[(NH₂-Gly)₄-Lys₂-Ser₂-Lys]_(n)-Ala-COOH, where n=1 to
 85. 29. Aconjugate according to claim 4 in which the pharmaceutically acceptablepolymer has the sequence of [(NH₂-X₀)₄-Lys₂-Y₂-Lys]_(n)-Z_(m)-COOH,where n=1 to 85; m=1 to 10; o=1 to 10; where X is any amino acid, whereY is any amino acid, and where Z is any amino acid.
 30. A conjugateaccording to claim 4 in which the pharmaceutically acceptable polymerhas the sequence of[(NH₂-Gly)₁₆-Lys₈-Lys₄-His₄-Glu₄-Lys₂-Lys]_(n)-Gly_(m)-Cys-COOH, wheren=1 to 85; where m=1 to
 10. 31. A conjugate according to claim 4 inwhich the pharmaceutically acceptable polymer has the sequence of[(NH₂-X)₁₆-Lys₈-Lys₄-Y₄-Z₄-Lys₂-Lys]_(n)-AA_(m)-Cys-COOH, where n=1 to85; where m=1 to 10; where X, Y, Z and AA represent any amino acidindependent of each other.
 32. A conjugate according to claim 4 whereinP is poly[N-(2-hydroxypropyl)-methacrylamide].
 33. A conjugate accordingto claim 1 or claim 4, wherein the biotin analogue selected fromiminobiotin, Biocytin hydrazide, Biotin hydrazide, biocytin,5-(Biotinamido)pentylamine, Sulfo-NHS(n-Hydroxysuccinimidyl)-Biotin,Sulfo-HNS-hexanyl-biotin (Sulfo-NHS-LD-Biotin), NHS-Biotin,Pentafluorophenyl-biotin, Pentafluorophenyl-polyethylenoxide-biotin,NHS-biotin Trifluoroacetamide, NHS-Iminobiotin trifluoroacetamide,Maleimido-polyethylenoxide biotin, Maleimido-polyethylenoxideiminobiotin, desthiobiotin, and chloracetyl-biotin.
 34. A conjugateaccording to claim 2, wherein the biotin or biotin analogue iselectrostatically or covalently linked to the polymer.
 35. A conjugateaccording to claim 3, wherein the biotin or biotin analogue physicallycoats a surface of the nanoparticle.
 36. A conjugate according to claim35, wherein the biotin or biotin analogue physically coats the surfaceof the nanoparticle via electrostatic bonding, hydrogen bonding orhydrophobic bonding.
 37. A conjugate according to claim 3, wherein thebiotin or biotin analogue is attached to the nanoparticle by covalentbonding.
 38. A conjugate according to claim 1, wherein the biotinanalogue has cytotoxic or anti-inflammatory activity.
 39. A process forsynthesising a polymeric conjugate, comprising one or more of thefollowing steps: a) reacting an active substance with a polymer to formsaid conjugate; b) chemically modifying the active substance to provideat least one functional group capable of forming a chemical linkage, andreacting the active substance and polymer to form said conjugate; c)chemically modifying a target molecule, which is biotin or an analoguethereof, to provide at least one functional group capable of forming achemical linkage and reacting the target molecule and polymer to formsaid conjugate; d) chemically modifying the active substance and thepolymer to provide functional groups capable of forming a chemicallinkage, and reacting the active substance and polymer to form saidconjugate; e) reacting the active substance with at least onecross-linking agent and reacting the active substance of polymer to formsaid conjugate; f) reacting the target molecule with at least onecross-linking agent and reacting the polymer and target molecule to formsaid conjugate; g) reacting the active substance and polymer with atleast one cross-linking agent and reacting the active substance andpolymer to form said conjugate; h) reacting the active substancedirectly with a polymeric support to form an intermediate containing oneor more molecules of the active substance linked to the polymer, andsubsequently coupling the polymer-active substance intermediate to oneor more target molecules; i) coupling one or more target molecules to apolymeric support and subsequently reacting the carrier-polymerintermediate with one or more molecules of the active substance to givea final conjugate containing one or more molecules of the activesubstance.
 40. A conjugate according to claim 1, wherein biotin or ananalogue thereof is a first targeting molecule, further comprising oneor more second targeting molecules, the second targeting moleculesperform a helper function for biotin-binding reactions necessary foruptake and/or transport of biotin in a cell.
 41. A conjugate accordingto claim 1, wherein biotin or an analogue thereof is a first targetingmolecule, further comprising one or more second targeting molecules,wherein the second targeting molecules assist in release of the activesubstance from the conjugate in a cell.
 42. A conjugate according toclaim 1, wherein biotin or an analogue thereof is a first targetingmolecule, further comprising one or more second targeting molecules,wherein the second targeting molecules promote a biological activity ofthe active substance.
 43. A process for the production of a conjugatehaving the general formula(B−Q)_(n)−P−(Q′−A)_(m) wherein B, Q, P, Q′, A, n and m are as defined inclaim 4, said process selected from any one or more of the followingsteps: a) reacting A with P to form an intermediate conjugate, andthereafter reacting the intermediate conjugate with biotin; b) reactingbiotin with P to form an intermediate conjugate and therafter reactingthe intermediate complex with A; c) the process of step a) or step b)wherein one or more of biotin, P or A are modified to provide at leastone functional group capable of forming a chemical linkage prior tocoupling with the other reactants; and d) reacting one or two of biotin,P or A with Q and/or Q′ prior to coupling with the other reactants. 44.A process according to claim 43 wherein Q and/or Q′ comprises anoptionally substituted alkylene C₁₋₅₀ moiety optionally within thechain, double bonds, triple bonds, aryl groups, and/or hetero atoms. 45.A process according to claim 43 wherein Q′ is a cleavable cross-linkingagent containing a disulfide bond.
 46. A process according to claim 45wherein the cross-linking agents are selected from disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl) suberate (BSS), ethyleneglycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (Sulfo-EGS), p-amino-phenylaceticacid, dithiobis(succinimidylpropionate) (DSP),3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidyltartarate (DST), disulfosuccinimidyl tartarate (Sulfo-DST),bis[2-(succinimidyloxycarbonyloxy)-ethylene]sulfone (BSOCOES),bis[2-(sulfosuccinimidooxycarbonyloxy)-ethylene]sulfone (Sulfo-BSOCOES),dimethyl adipimidate.2 HCl (DMA), dimethyl pimelimidate.2 HCl (DMP),dimethyl suberimidate.2 HCl (DMS).
 47. A process according to claim 43wherein said spacer is selected from disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BSS), ethyleneglycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (Sulfo-EGS), p-amino-phenylaceticacid, dithiobis(succinimidylpropionate) (DSP),3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidyltartarate (DST), disulfosuccinimidyl tartarate (Sulfo-DST),bis[2-(succinimidyloxycarbonyloxy)-ethylene]sulfone (BSOCOES),bis[2-(sulfosuccinimidooxycarbonyloxy)-ethylene]sulfone (Sulfo-BSOCOES),dimethyl adipimidate.2 HCl (DMA), dimethyl pimelimidate.2 HCl (DMP),dimethyl suberimidate.2 HCl (DMS).
 48. A process according to claim 43wherein said spacer is selected from N-succinimidyl3-(2-pyridyldithio)propionate (SPDP), iminothiolane, sulfosuccinimidyl6-[3-(2-pyridyldithio) propionamido]hexanoate (Sulfo-LC-SPDP),succinimidyl 6-[3-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP),sulfosuccinimidyl 6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate(Sulfo-LC-SMPT), 1,4-di[3′-(2′-pyridyldithio)propionamido]butane(DPDPB), 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene(SMPT), dimethyl 3,3′dithiobispropionimidate.2 HCl (DTBP).
 49. A processaccording to claim 45 wherein the cross-linking agents are selected fromN-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), iminothiolane,sulfosuccinimidyl 6-[3-(2-pyridyldithio) propionamido]hexanoate(Sulfo-LC-SPDP), succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP), sulfosuccinimidyl6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate (Sulfo-LC-SMPT),1,4-di[3′-(2′-pyridyldithio)propionamido]butane (DPDPB),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT),dimethyl 3,3′dithiobispropionimidate.2 HCl (DTBP).
 50. A conjugateprepared by a process of claim
 43. 51. A conjugate of claim 3, whereinthe nanoparticle is prepared by solvent evaporation, complexcoacervation, polymer/polymer incompatibility, gelation, interfacialpolymerisation or thermal denaturation.
 52. A conjugate of claim 3,wherein the nanoparticle is biodegradable.
 53. A process for theproduction of a conjugate of claim 52, which process comprises one ormore of the following steps: a) reacting nanospheres with a targetingmolecule to form the conjugate; b) chemically modifying a targetingmolecule to provide at least one functional group capable of forming achemical linkage and reacting nanospheres and the modified targetingmolecules to form the conjugate; c) reacting nanospheres with at leastone cross-linking agent to prepare “activated” nanoparticles which arereacted with a targeting molecule to form the conjugate; d) reacting atargeting molecule with at least one cross-linking agent and reactingthe nanospheres with the reacted targeting molecule to form theconjugate; e) reacting nanospheres and a targeting molecule with atleast one cross-linking agent to the conjugate; f) reacting nanosphereswith at least one cross-linking agent, reacting a targeting moleculewith at least one cross-linking agent and reacting the reactednanospheres and the reacted targeting molecule to form the conjugate; org) reacting a targeting molecule with at least one cross-linking agentto prepare an analogue which is reacted with a hydrophobic moiety toform a hydrophobic derivative of the targeting molecule, and thenincubating the hydrophobic derivative of the targeting molecule with ananosphere in such a manner that the nanosphere is coatedhydrophobically with the targeting molecule.
 54. A process of claim 53,wherein the cross-linking agent contains a disulfide bond or iscleavable by acid, base or periodate.
 55. A process of claim 53, whereinthe cross-linking agent is selected from the group consisting ofN-(4-azidophenylthio)phthalimide, 4,4′-dithiobisphenylazide,dithiobis(succinimidylpropionate),dimethyl-3,3′-dithiobispropionimidate.2HCl,3,3′-dithiobis-(sulfosuccinimidylpropionate),ethyl-4-azidophenyl)-1,3′dithiopropionate,sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiobutyrimidate.HCl,N-succinimidyl-(4-azidophenyl)-1,3′dithiopropionate;sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate,sulfosuccinimidyl-2-(p-azidosalicylamido)-ethyl-1,3′-dithiopropionate,N-succinimidyl-3-(2-pyridylthio)propionate,sulfosuccinimidyl-(4-azidophenyldithio)-propionate, 2-iminothiolane,disuccinimidyl tartrate andbis-[2-(succinimidyloxycarbonyloxy)ethyl]-sulfone.
 56. A process ofclaim 53, wherein the targeting molecule is cross-linked to thenanosphere or nanoparticle by reaction of the carrier with acarbodiimide and N-hydroxysuccinimide (NHS), and then reacting the NHSderivative with a suitable functional group on the nanosphere.
 57. Aprocess of claim 53, wherein the cross-linking agent contains abiodegradable bond.
 58. A process of claim 57, wherein the cross-linkingagent is cleaved by an esterase, glutathione, or azo-reductase.
 59. Aconjugate prepared by a process of claim
 53. 60. A method for themodification of a polymeric support to introduce functional groupscapable of reacting either directly with an active substance or with achemically-modified form of the active substance, wherein a resultingpolymer-active substance intermediate contains one or more molecules ofthe active substance, said intermediate being suitable for coupling tobiotin or an analogue thereof to give a conjugate capable of amplifieddelivery of the active substance.
 61. A pharmaceutical composition whichcomprises a conjugate according to any one of claims 1 to 21, 24 or28-38 together with a pharmaceutically acceptable carrier or excipient.62. A diagnostic imaging composition comprising a conjugate according toany one of claims 22 to
 27. 63. A method for the treatment orprophylaxis of disease which comprises administering to a subject atherapeutically effective amount of a conjugate according to any one ofclaims 1 to 21, 24 or claim 28-38 or a composition of claim
 61. 64. Amethod of claim 63 wherein the disease is cancer.
 65. A method of claim63, wherein the disease is an inflammatory condition.
 66. A method ofclaim 65, wherein the disease is rheumatoid arthritis.
 67. A method ofclaim 65, wherein the disease is Crohn's disease.
 68. A method of claim65, wherein the disease is inflammatory bowel disease.
 69. A method ofclaim 63, wherein the disease is multiple sclerosis.
 70. Use of aconjugate according to any one of claims 1 to 21, 24, or 24 to 28 in themanufacture of a medicament.
 71. Use of a conjugate according to any oneof claims 22 to 27 in the manufacture of a diagnostic imaging agent. 72.A method for the diagnosis of a pathological condition which comprisesadministering to a subject an effective amount of a conjugate accordingto claim 22 to 27 or a composition according to claim
 62. 73. Aconjugate according to claim 4, wherein the linker is biodegradable. 74.A conjugate according to claim 4, wherein the linker is a hydrazone. 75.A conjugate according to claim 4, wherein the linker contains5-benzoyl-valeric acid.
 76. A conjugate according to claim 4, whereinthe linker is biodegradable and contains a valine-citrilline dipeptide.77. A conjugate according to claim 4, wherein the linker isbiodegradable and contains a phenylalanine-lysine dipeptide.
 78. Aconjugate of claim 1, wherein the active substance is a drug selectedfrom platinum derivatives.
 79. A conjugate according to claim 78,wherein the platinum derivative is selected from cis-Platin,CarboPlatin, oxaliplatin, multinuclear platinate species includingBBR3464 and BBR3005, transdiamminedichloroplatinum (II) (Transplatin),chlorodiethylenetriammineplatinum (II), Platinum IV compounds,spiroplatin, platin-phosphine derivatives.
 80. A conjugate of claim 1,wherein the active substance is doxorubicin or an analogue thereof,including daunorubicin, daunomycin, epirubicin, adriamycin.
 81. Aconjugate of claim 1, wherein the active agent is a cytotoxin selectedfrom anti-folates including methotrexate and dichloromethatrexate.
 82. Aconjugate of claim 1, wherein the biotin is a hydrazidyl derivative ofbiotin.
 83. A conjugate of claim 1, wherein the biotin is chloracetylbiotin.
 84. A conjugate of claim 1, wherein the biotin is desthiobiotin.85. A conjugate of claim 79, wherein the biotin is desthiobiotin.
 86. Aconjugate of claim 1, wherein the active substance is a dolastatinderivative.
 87. A conjugate of claim 86, wherein the dolastatinderivative is auristatin or monomethylauristatin.
 88. A conjugate ofclaim 4, wherein the linker is a valine-citrilline-aminobenzyl-carbamatederivative.